Investigation of toxicity and mutagenicity of cold atmospheric argon plasma.

Cold atmospheric argon plasma is recognized as a new contact free approach for the decrease of bacterial load on chronic wounds in patients. So far very limited data are available on its toxicity and mutagenicity on eukaryotic cells. Thus, the toxic/mutagenic potential of cold atmospheric argon plasma using the MicroPlaSter ß® , which has been used efficiently in humans treating chronic and acute wounds, was investigated using the XTT assay in keratinocytes and fibroblasts and the HGPRT (hypoxanthine guanine phosphoribosyl transferase) assay with V79 Chinese hamster cells. The tested clinical parameter of a 2 min cold atmospheric argon plasma treatment revealed no relevant toxicity on keratinocytes (viability: 76% ± 0.17%) and on fibroblasts (viability: 81.8 ± 0.10) after 72 hr as compared to the untreated controls. No mutagenicity was detected in the HGPRT assay with V79 cells even after repetitive CAP treatments of 2-10 min every 24 hr for up to 5 days. In contrast, UV-C irradiation of V79 cells, used as a positive control in the HGPRT test, led to DNA damage and mutagenic effects. Our findings indicate that cold atmospheric plasma using the MicroPlaSter ß® shows negligible effects on keratinocytes and fibroblasts but no mutagenic potential in the HGPRT assay, indicating a new contact free safe technology. Environ. Mol. Mutagen. 58:172-177, 2017. © 2017 Wiley Periodicals, Inc.

Investigation of the mutagenic potential of cold atmospheric plasma at bactericidal dosages.

In the past few years, cold atmospheric plasma (CAP) has evolved into a new tool in the fight against nosocomial infections and antibiotic-resistant microorganisms. The products generated by the plasma-electrons, ions, reactive species and UV light-represent a 'lethal cocktail' for different kinds of pathogen, which opens up possible applications in hygiene and medicine. Nevertheless, to ensure the safe usage of CAP on skin (e.g., to treat wounds or skin diseases) several pre-clinical in vitro studies have to be performed before implementing clinical trials on humans. In the study presented here, inactivation experiments with Escherichia coli were carried out to identify the necessary plasma dosage for a 5 log reduction: with a small hand-held battery-operated CAP device, these disinfection properties were achieved after application during 30s. This and higher plasma dosages were then used to analyze the mutagenicity induced in V79 Chinese hamster cells-to furthermore define a 'safe application window'-with the HPRT (hypoxanthine-guanine phosphoribosyl transferase) mutation assay. The results show that a CAP treatment of up to 240 s and repeated treatments of 30s every 12h did not induce mutagenicity at the Hprt locus beyond naturally occurring spontaneous mutations.

A randomized two-sided placebo-controlled study on the efficacy and safety of atmospheric non-thermal argon plasma for pruritus.

BACKGROUND: To look into new potential indications for physical plasma and because some reports suggest plasma having antipruritic effects, we investigated the treatment of pruritus that often represents a therapeutic challenge. OBJECTIVES: To assess the efficacy and safety of cold atmospheric argon plasma as add-on-therapy in pruritic diseases. METHODS: We treated 46 patients with various pruritic diseases with cold plasma for 2 min daily in addition to standard treatment. All patients served as their own control, when their pruritic disease was treated with argon gas (placebo). The outcome measure was a long-term and short-term reduction in itching measured by means of a visual analogue score (VAS). RESULTS: The VAS scores at baseline were comparable (plasma 4.57, SD 2.38, argon 4.34, SD 2.35). We did not find any significant differences in VAS reduction between plasma and argon: long-term VAS difference of 1.97 (SD 1.33) for plasma and 1.74 (SD 2.37) for argon [P = 0.224, 95% CI: (-0.15; 0.60)], short-term VAS difference of 1.92 (SD 1.33) for plasma and 1.97 (SD 1.29) for argon [P = 0.544, 95% CI: (-0.21; 0.11)]. In both groups, patients experienced a significant reduction of pruritus at the end of therapy compared to baseline [plasma 1.97 (P < 0.0001), placebo 1.74 [P < 0.0001)]. No relevant side effects occurred, and treatment was well tolerated. CONCLUSIONS: Treatment with cold plasma did not result in higher pruritus reduction than treatment with placebo. A significant reduction of pruritus compared to no effect was found at the end of therapy in both groups. Both treatment options had similar safety profiles.

Cold atmospheric plasma for local infection control and subsequent pain reduction in a patient with chronic post-operative ear infection.

Following surgery of cholesteatoma, a patient developed a chronic infection of the external auditory canal, including extended-spectrum ß-lactamase producing Escherichia coli, which caused severe pain. The application of cold atmospheric plasma resulted in a significant reduction in pain and clearance of bacterial carriage, allowing antibiotics and analgesics to be ceased.

Successful and safe use of 2 min cold atmospheric argon plasma in chronic wounds: results of a randomized controlled trial.

BACKGROUND: The development of antibiotic resistance by microorganisms is an increasing problem in medicine. In chronic wounds, bacterial colonization is associated with impaired healing. Cold atmospheric plasma is an innovative promising tool to deal with these problems. OBJECTIVES: The 5-min argon plasma treatment has already demonstrated efficacy in reducing bacterial numbers in chronic infected wounds in vivo. In this study we investigated a 2-min plasma treatment with the same device and the next-generation device, to assess safety and reduction in bacterial load, regardless of the kind of bacteria and their resistance level in chronic wounds. METHODS: Twenty-four patients with chronic infected wounds were treated in a prospective randomized controlled phase II study with 2 min of cold atmospheric argon plasma every day: 14 with MicroPlaSter alpha device, 10 with MicroPlaSter beta device (next-generation device) in addition to standard wound care. The patient acted as his/her own control. Bacterial species were detected by standard bacterial swabs and bacterial load by semiquantitative count on nitrocellulose filters. The plasma settings were the same as in the previous phase II study in which wounds were exposed for 5 min to argon plasma. RESULTS: Analysis of 70 treatments in 14 patients with the MicroPlaSter alpha device revealed a significant (40%, P<0.016) reduction in bacterial load in plasma-treated wounds, regardless of the species of bacteria. Analysis of 137 treatments in 10 patients with the MicroPlaSter beta device showed a highly significant reduction (23.5%, P<0.008) in bacterial load. No side-effects occurred and the treatment was well tolerated. CONCLUSIONS: A 2-min treatment with either of two cold atmospheric argon plasma devices is a safe, painless and effective technique to decrease the bacterial load in chronic wounds.

A first prospective randomized controlled trial to decrease bacterial load using cold atmospheric argon plasma on chronic wounds in patients.

BACKGROUND: Bacterial colonization of chronic wounds slows healing. Cold atmospheric plasma has been shown in vitro to kill a wide range of pathogenic bacteria. Objectives To examine the safety and efficiency of cold atmospheric argon plasma to decrease bacterial load as a new medical treatment for chronic wounds. PATIENTS AND METHODS: Thirty-eight chronic infected wounds in 36 patients were treated in a prospective randomized controlled phase II study with 5 min daily cold atmospheric argon plasma in addition to standard wound care. The patient acted as his or her own control. Bacterial species were detected by standard bacterial swabs and semiquantitative changes by nitrocellulose filters. Plasma setting and safety had been determined in a preceding phase I study. RESULTS: Analysis of 291 treatments in 38 wounds found a highly significant (34%, P < 10(-6)) reduction of bacterial load in treated wounds, regardless of the type of bacteria. No side-effects occurred and the treatment was well tolerated. CONCLUSIONS: Cold atmospheric argon plasma treatment is potentially a safe and painless new technique to decrease bacterial load of chronic wounds and promote healing.

N-hydroxyguanidines as new heme ligands: UV-visible, EPR, and resonance Raman studies of the interaction of various compounds bearing a C=NOH function with microperoxidase-8.

Interaction between microperoxidase-8 (MP8), a water-soluble hemeprotein model, and a wide range of N-aryl and N-alkyl N'-hydroxyguanidines and related compounds has been investigated using UV-visible, EPR, and resonance Raman spectroscopies. All the N-hydroxyguanidines studied bind to the ferric form of MP8 with formation of stable low-spin iron(III) complexes characterized by absorption maxima at 405, 535, and 560 nm. The complex obtained with N-(4-methoxyphenyl) N'-hydroxyguanidine exhibits EPR g-values at 2.55, 2.26, and 1.86. The resonance Raman (RR) spectrum of this complex is also in agreement with an hexacoordinated low-spin iron(III) structure. The dissociation constants (K(s)) of the MP8 complexes with mono- and disubstituted N-hydroxyguanidines vary between 15 and 160 microM at pH 7.4. Amidoximes also form low-spin iron(III) complexes of MP8, although with much larger dissociation constants. Under the same conditions, ketoximes, aldoximes, methoxyguanidines, and guanidines completely fail to form such complexes with MP8. The K(s) values of the MP8-N-hydroxyguanidine complexes decrease as the pH of the solution is increased, and the affinity of the N-hydroxyguanidines toward MP8 increases with the pK(a) of these ligands. Altogether these results show that compounds involving a -C(NHR)=NOH moiety act as good ligands of MP8-Fe(III) with an affinity that depends on the electron-richness of this moiety. The analysis of the EPR spectrum of the MP8-N-hydroxyguanidine complexes according to Taylor's equations shows a strong axial distortion of the iron, typical of those observed for hexacoordinated heme-Fe(III) complexes with at least one pi donor axial ligand (HO(-), RO(-), or RS(-)). These data strongly suggest that N-hydroxyguanidines bind to MP8 iron via their oxygen atom after deprotonation or weakening of their O-H bond. It thus seems that N-hydroxyguanidines could constitute a new class of strong ligands for hemeproteins and iron(III)-porphyrins.

Evidence for changes in the nucleotide conformation in the active site of H(+)-ATPase as determined by pulsed EPR spectroscopy.

The conformation of di- and triphosphate nucleosides in the active site of ATPsynthase (H(+)-ATPase) from thermophilic Bacillus PS3 (TF1) and their interaction with Mg(2+)/Mn(2+) cations have been investigated using EPR, ESEEM, and HYSCORE spectroscopies. For a ternary complex formed by a stoichiometric mixture of TF1, Mn(2+), and ADP, the ESEEM and HYSCORE data reveal a (31)P hyperfine interaction with Mn(2+) (|A((31)P)| approximately 5.20 MHz), significantly larger than that measured for the complex formed by Mn(2+) and ADP in solution (|A((31)P)| approximately 4.50 MHz). The Q-band EPR spectrum of the Mn.TF1.ADP complex indicates that the Mn(2+) binds in a slightly distorted environment with |D| approximately 180 x 10(-4) cm(-1) and |E| approximately 50 x 10(-4) cm(-1). The increased hyperfine coupling with (31)P in the presence of TF1 reflects the specific interaction between the central Mn(2+) and the ADP beta-phosphate, illustrating the role of the enzyme active site in positioning the phosphate chain of the substrate for efficient catalysis. Results with the ternary Mn.TF1.ATP and Mn.TF1.AMP-PNP complexes are interpreted in a similar way with two hyperfine couplings being resolved for each complex (|A((31)P(beta))| approximately 4.60 MHz and |A((31)P(gamma))| approximately 5.90 MHz with ATP, and |A((31)P(beta))| approximately 4.20 MHz and |A((31)P(gamma))| approximately 5.40 MHz with AMP-PNP). In these complexes, the increased hyperfine coupling with (31)P(gamma) compared with (31)P(beta) reflects the smaller Mn.P distance with the gamma-phosphate compared with the beta-phosphate as found in the crystal structure of the analogous enzyme from mitochondria [3.53 vs 3.70 A (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628)] and the different binding modes of the two phosphate groups. The ESEEM and HYSCORE data of a complex formed with Mn(2+), ATP, and the isolated beta subunit show that the (31)P hyperfine coupling is close to that measured in the absence of the protein, indicating a poorly structured nucleotide site in the isolated beta subunit in the presence of ATP. The inhibition data obtained for TF1 incubated in the presence of Mg(2+), ADP, Al(NO(3))(3), and NaF indicate the formation of the inhibited complex with the transition state analogue namely Mg.TF1.ADP.AlF(x) with the equilibrium dissociation constant K(D) = 350 microM and rate constant k = 0.02 min(-1). The ESEEM and HYSCORE data obtained for an inhibited TF1 sample, Mn.TF1.ADP.AlF(x), confirm the formation of the transition state analogue with distinct spectroscopic footprints that can be assigned to Mn.(19)F and Mn.(27)Al hyperfine interactions. The (31)P(beta) hyperfine coupling that is measured in the inhibited complex with the transition state analogue (|A((31)P(beta))| approximately 5.10 MHz) is intermediate between those measured in the presence of ADP and ATP and suggests an increase in the bond between Mn and the P(beta) from ADP upon formation of the transition state.

The role of the Mg2+ cation in ATPsynthase studied by electron paramagnetic resonance using VO2+ and Mn2+ paramagnetic probes.

The electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectra of Mg2+-depleted chloroplast F1-ATPase substituted with stoichiometric VO2+ are reported. The ESEEM and HYSCORE spectra of the complex are dominated by the hyperfine and quadrupole interactions between the VO2+ paramagnet and two different nitrogen ligands with isotropic hyperfine couplings /A1/ = 4.11 MHz and /A2/ = 6.46 MHz and nuclear quadrupole couplings e2qQ1 approximately 3.89-4.49 MHz and e2qQ2 approximately 1.91-2.20 MHz, respectively. Aminoacid functional groups compatible with these magnetic couplings include a histidine imidazole, the epsilon-NH2 of a lysine residue, and the guanidinium group of an arginine. Consistent with this interpretation, very characteristic correlations are detected in the HYSCORE spectra between the 14N deltaM1 = 2 transitions in the negative quadrant, and also between some of the deltaM1 = 1 transitions in the positive quadrant. The interaction of the substrate and product ADP and ATP nucleotides with the enzyme has been studied in protein complexes where Mg2+ is substituted for Mn2+. Stoichiometric complexes of Mn x ADP and Mn x ATP with the whole enzyme show distinct and specific hyperfine couplings with the 31P atoms of the bonding phosphates in the HYSCORE (ADP, A(31Pbeta) = 5.20 MHz: ATP, A(31Pbeta) = 4.60 MHz and A(31Pgamma) = 5.90 MHz) demonstrating the role of the enzyme active site in positioning the di- or triphosphate chain of the nucleotide for efficient catalysis. When the complexes are formed with the isolated alpha or beta subunits of the enzyme, the HYSCORE spectra are substantially modified, suggesting that in these cases the nucleotide binding site is only partially structured.

Identification and characterization of Mg2+ binding sites in isolated alpha and beta subunits of H(+)-ATPase from Bacillus PS3.

The properties of the nucleotide binding sites in the isolated beta and alpha subunits of H(+)-ATPase from Bacillus PS3 (TF1) have been examined by studying the EPR properties of bound VO(2+), which is a paramagnetic probe for the native Mg2+ cation cofactor. The amino acid ligands of the VO2+ complexes with the isolated beta subunit, with the isolated alpha subunit, with different mixtures of both alpha and beta subunits, and with the catalytic alpha 3 beta 3 gamma subcomplex have been characterized by a combination of EPR, ESEEM, and HYSCORE spectroscopies. The EPR spectrum of the isolated beta subunit with bound VO2+ (1 VO2+/beta) is characterized by (51)V hyperfine coupling parameters (A( parallel) = 168 x 10(-)(4) cm(-)(1) and A( perpendicular) = 60 x 10(-)(4) cm(-)(1)) that suggest that VO2+ binds to the isolated beta subunit with at least one nitrogen ligand. Results obtained for the analogous VO2+ complex with the isolated alpha subunit are virtually identical. ESEEM and HYSCORE spectra are also reported and are similar for both complexes, indicating a very similar coordination scheme for VO2+ bound to isolated alpha and beta subunits. In the isolated beta (or alpha) subunit, the bound VO2+ cation is coordinated by one nitrogen ligand with hyperfine coupling parameters A( parallel)((14)N) = 4.44 MHz, and A( perpendicular)((14)N) = 4.3 MHz and quadrupole coupling parameters e(2)()qQ approximately 3.18 MHz and eta approximately 1. These are typical for amine-type nitrogen ligands equatorial to the VO2+ cation; amino acid residues in the TF1 beta and alpha subunits with nitrogen donors that may bind VO2+ are reviewed. VO2+ bound to a mixture of alpha and beta subunits in the presence of 200 mM Na2SO4 to promote the formation of the alpha 3 beta 3 hexamer has a second nitrogen ligand with magnetic properties similar to those of a histidine imidazole. This situation is analogous to that in the alpha 3 beta 3 gamma subcomplex and in the whole TF1 enzyme [Buy, C., Matsui, T., Andrianambinintsoa, S., Sigalat, C., Girault, G., and Zimmermann, J.-L. (1996) Biochemistry 35, 14281-14293]. These data are interpreted in terms of only partially structured nucleotide binding sites in the isolated beta and alpha subunits as compared to fully structured nucleotide binding sites in the alpha 3 beta 3 heterohexamer, the alpha 3 beta 3 gamma subcomplex, and the whole TF1 ATPase.

Cooperativity between the enzymatic sites of F1-ATPase revisited by the use of HPLC methods.

The fundamental question of the cooperativity between the enzymatic sites of F1-ATPase is examined in the light of new measurements of the enzymatic rate of ATP hydrolysis by CF1, the enzyme isolated from spinach chloroplasts. The experimental data, obtained with a chromatographic method, fit a model that involves two kinds of independent enzymatic sites working with metal-free ATP, with no need of cooperativity between the sites. Binding measurements between ADP or ATP and CF1 by the chromatographic method of Hummel and Dreyer (1962) also support this conclusion. The present data and interpretation are in agreement with those reported recently (Reynafarje and Pedersen, 1996) which show that the first order rate constant of ATP hydrolysis by MF1, the analogous enzyme from mitochondria, is virtually constant under experimental conditions involving either unisite or multisite hydrolysis of ATP. The present data and interpretation are discussed together with those reported previously, in particular with regard to the methods that were used to support the commonly accepted opposite viewpoint.

Binding sites for Mg(II) in H(+)-ATPase from Bacillus PS3 and in the alpha 3 beta 3 gamma subcomplex studied by one-dimensional ESEEM and two-dimensional HYSCORE spectroscopy of oxovanadium(IV) complexes: a possible role for beta-His-324.

The binding sites for Mg2+ in wild type F1 ATPase (TF1) and in the alpha 3 beta 3 gamma subcomplex from the thermophilic bacterium Bacillus PS3 have been studied by EPR and by ESEEM and HYSCORE spectroscopy of complexes with the oxovanadium cation VO2+. Complexes of metal-depleted TF1 and substoichiometric amounts of VO2+ display low-temperature EPR signals with spectral parameters g parallel = 1.947 and g perpendicular = 1.980, and hyperfine couplings with 51V, A parallel = 169 x 10(-4) cm-1 and A perpendicular = 61 x 10(-4) cm-1, that are indicative of a binding site for VO2+ with nitrogen ligands from the protein. This binding site is probably identical with the metal binding site with strong affinity M1 that has been characterized using Mn2+ in a previous study [Buy, C., Girault, G., & Zimmermann, J. L. (1996) Biochemistry 35, 9880-9891]. The three-pulse ESEEM spectrum of the VO2+ complex with TF1 shows a frequency pattern with spectral properties that are evidence for two nitrogen ligands to the VO2+ with hyperfine couplings A1 = 4.75 MHz and A2 = 6.5 MHz and nuclear quadrupole parameters e2Qq1 = 2.8-3.2 MHz and e2Qq2 = 2.0-2.3 MHz. The ligands are identified as a lysine terminal amine and a histidine imidazole, which are proposed as Lys-164 and His-324 from a beta subunit. The HYSCORE data obtained for the VO.TF1 complex show correlations within each pair of the ESEEM nu dq peaks from the 14N nuclei, confirming the interpretation of the one-dimensional spectra. Evidence for the formation of a ternary complex by addition of VO2+ and ATP to metal-depleted TF1 is shown in the EPR and ESEEM spectra and in the contour plots of the HYSCORE data. Two pairs of correlation patterns are resolved in addition to the peaks from the two 14N ligands, which are interpreted as hyperfine couplings with 31P beta and 31P gamma of the ATP that binds the VO2+ cation. The assignment of the two hyperfine couplings to the specific phosphates, A(31P beta) = 15.5 MHz and A(31P gamma) = 8.7 MHz, in the VO.TF1.ATP complex is proposed by comparison with those measured for VO2+ in solution with ATP at pH 6.3 and 2.3. These results are discussed in light of the previous data with the analogous Mn.TF1 complex, and a model is proposed in which the native Mg2+ in the M1 site is coordinated by the side chain of beta-Lys-164 and is in close proximity to a histidine residue (probably beta-His-324) that may have a critical role. Additional coordination by two phosphates from ATP (probably the beta- and gamma-phosphates) is observed in the ternary complex VO.TF1.ATP. ESEEM and HYSCORE data are also obtained for the analogous complexes VO. alpha 3 beta 3 gamma and VO. alpha 3 beta 3 gamma .ATP that show very similar properties in terms of coordination of the divalent metal cation, except for the lysine ligand that is found to be lost in the ternary complex with ATP. It is suggested that this observation may reflect changes in the metal and nucleotide active sites that are associated with the absence of the delta and epsilon subunits in the subcomplex.

Novel acid labile COL1 trityl-linked difluoronucleoside immunoconjugates: synthesis, characterization, and biological activity.

LY207702 (1) is a difluorinated purine nucleoside that exhibits impressive antitumor activity in preclinical models. This agent, however, also possesses cardiotoxicity which limits the potential clinical utility of this novel drug candidate. We therefore developed linker chemistry whereby regioselective N6-tritylation of LY207702 (1) allowed this drug to be coupled to epsilon-lysine amino groups of mAb's reactive with human tumor-associated antigens. The resulting immunoconjugates 3 possessed conjugation ratios ranging from 5 to 7 mol of LY207702/mol of mAb, minimal aggregate content (5-10%), and good immunoreactivity. The electronic nature of substituents on the aromatic rings of the trityl group dictated the degree of acid lability of the trityl linker. Increased electronic stabilization of the transient trityl carbocation led to increase in the release rate of free drug, i.e., m-DMT 10a = p-DMT 10b > p-MMT 10d > p-T 10f. Consequently, the more acid labile DMT conjugates 3a and 3b proved to be the most potent cytotoxic agents, and the most stable p-T conjugate 3f exhibited the least antitumor activity when evaluated in vitro and in vivo. p-MeT-linked conjugate 3e, the most stable construct that retained excellent in vivo antitumor activity, was selected for more extensive evaluation. No detectable free drug or metabolite was observed in mouse plasma at a single intravenous dose of p-MeT conjugate 3e, which was consistent with its predicted stability under physiological conditions. This construct did, however, exhibit significant antigen-mediated antitumor activity in vivo. No cardiotoxicity was detected in mice dosed with conjugate 3e (6 mg/kg free drug content per day for 21 days) equivalent to approximately 8 times the total dose required for complete regression of well-established (approximately 1 g) HC1 human colon tumor xenografts in nude mice. Cardiotoxicity was induced in 20% of free drug 1 treated group at the equivalent dose. Cardiomyopathy was, however, observed when the dose of conjugate 3e was increased to 8 mg/kg per day for 21 days. These data suggest that antitumor activity of LY207702 (1) was maintained and its cardiotoxic potential reduced when this agent was administered to human tumor xenograft bearing nude mice as COL1-N6-p-MeT-207702 conjugate 3e.

Metal binding sites of H(+)-ATPase from chloroplast and Bacillus PS3 studied by EPR and pulsed EPR spectroscopy of bound manganese(II).

The metal binding sites of isolated F1 ATPase from spinach chloroplasts (CF1) and from the thermophilic bacterium Bacillus PS3 (TF1) have been studied by EPR and pulsed EPR spectroscopy using Mn(II) as a paramagnetic probe. After dialysis in the presence of EDTA, purified CF1 retains 0.14 +/- 0.07 Mg(II) and approximately 0.75 +/- 0.25 ADP. TF1 retains 0.31 +/- 0.03 Mg(II) and 0.08 +/- 0.01 nucleotide (ADP + ATP) after the same treatment. Supplementing known quantities of Mn(II) to metal-depleted CF1 allowed a spectroscopic characterization of the bound Mn(II) cations, for which the EPR spectra at X- and Q-band are reported. The zero field splitting parameters of Mn(II) are derived from the simulation of the EPR signal recorded at Q-band for a sample supplemented with 0.3 Mn/CF1. The values, magnitude of D approximately 200 x 10(-4) cm-1 and magnitude of E approximately 40 x 10(-4) cm-1 suggest that the Mn(II) binds to CF1 in a slightly distorted environment. The ESEEM spectra of complexes of Mn(II) with CF1 were also recorded for different Mn/CF1 ratios. For a complex with 0.8 Mn/CF1, the ESEEM spectrum shows two frequencies at 3.7 and 8.6 MHz that are attributed to the magnetic coupling with 31P with a hyperfine coupling constant of magnitude of A approximately 5.3 MHz, reflecting the interaction with a phosphate group from the endogenous ADP molecule. This demonstrates close proximity of the strong affinity metal site M1 and the endogenous ADP binding site N1, and binding of the ADP beta-phosphate to the divalent metal cation. For Mn(II) complexes with higher Mn/CF1 ratios, new frequency components below approximately 5 MHz are resolved in the spectra in addition to the peaks from 31P. From a comparison of the CF1 spectra and their magnetic field dependence across the Mn(II) EPR line shape with those of Mn(II) complexes with imidazole, glycine, poly-L-lysine, and nucleotide ligands, it is concluded that additional metal binding sites are filled at higher Mn contents and that these involve 14N donors. It is suggested that the most probable set of ligands of the divalent metal(s) for these additional metal sites in CF1 includes a lysine residue, in line with a previous proposal [Houseman, A. L. P., Morgan, L., LoBrutto, R., & Frasch, W. D. (1994) Biochemistry 33, 4910-4917]. Similar experiments for a Mn(II) complex with TF1 (0.4 Mn/TF1) showed no interaction with 31P; instead modulations are detected in the ESEEM below approximately 5 MHz that are attributed to a 14N ligand. This is tentatively attributed to the deprotonated amine of Lys-162 from a beta subunit, on the basis of the structural data available for the mitochondrial F1 complex. Addition of the substrate ATP to this Mn.TF1 complex leads to the formation of a ternary Mn.TF1.ATP complex with coordination of the Mn(II) by a phosphate group from the ATP as judged from the ESEEM results (magnitude of A(31P) approximately 4.5 MHz). An increase in the hyperfine coupling constant of 31P of the phosphate bound to Mn(II) to magnitude of A(31P) approximately 5.1 MHz is observed after incubation of the ternary complex at room temperature. This is interpreted as a significant rearrangement of the coordination sphere of the Mn(II) in the M1 site of the Mn.TF1.ATP complex and may reflect conformational changes of catalytic significance that occur in the nucleotide binding site during unisite hydrolysis of ATP to ADP by this complex.

General pharmacology of insulin lispro in animals.

[Lys(B28),Pro(B29)]-human insulin (insulin lispro, CAS 133107-64-9, LY275585, Humalog) is a quick acting insulin analog which is currently undergoing clinical evaluation for the treatment of diabetes. The potential secondary pharmacological activity of insulin lispro was profiled in studies for the evaluation of effects on the central and autonomic nervous system, the cardiovascular system, urine and electrolyte excretion, and gastrointestinal function. In vivo doses ranged from 0.03 to 10 U/kg, administered by the subcutaneous route, while pharmacologic activity in vitro was examined in smooth and cardiac muscle at concentrations of 1 x 10(-9) to 1 x 10(-5) mol/l. Insulin lispro exhibited secondary pharmacological activity in central nervous system tests only at higher doses with the most prominent observations being sedation and decreased responsiveness. Insulin lispro was essentially inactive in tests of autonomic (smooth and cardiac muscle), cardiovascular (mean arterial pressure, heart rate, systolic pressure, diastolic pressure, and pulse pressure), renal (urine and electrolyte excretion) and gastrointestinal (motility) function. In summary, insulin lispro had minimal effect in these pharmacodynamic studies indicating that insulin lispro has minimal potential to produce adverse pharmacological side effects at clinically relevant doses.

EPR, electron spin echo envelope modulation, and electron nuclear double resonance studies of the 2Fe2S centers of the 2-halobenzoate 1,2-dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS.

The 2-halobenzoate 1,2-dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS (Fetzner, S., Müller, R., and Lingens, F. (1992) J. Bacteriol. 174, 279-290) contains both a ferredoxin-type and a Rieske-type 2Fe2S center. These two significantly different 2Fe2S clusters were characterized with respect to their EPR spectra, electrochemical properties (Rieske-type cluster with gz = 2.025, gy = 1.91, gx = 1.79, gav = 1.91, Em = -125 +/- 10 mV; ferredoxin-type center with gz = 2.05, gy = 1.96, gx = 1.89, gav = 1.97, Em = -200 +/- 10 mV) and pH dependence thereof. X band electron spin echo envelope modulation and electron nuclear double resonance spectroscopy was applied to study the interaction of the Rieske-type center of the 2-halobenzoate 1,2-dioxygenase with 14N and 1H nuclei in the vicinity of the 2Fe2S cluster. The results are compared to those obtained on the Rieske protein of the cytochrome b6f complex (Em = +320 mV) and the water-soluble ferredoxin (Em = -430 mV) of spinach chloroplasts, as typical representatives of the gav = 1.91 and gav = 1.96 class of 2Fe2S centers. Properties common to all Rieske-type clusters and those restricted to the respective centers in bacterial oxygenases are discussed.

Pulsed EPR studies of the binuclear Mn(III)Mn(IV) center in catalase from Thermus thermophilus.

The nature of possible protein ligands to the binuclear metal core in manganese catalase from Thermus thermophilus has been addressed by EPR and ESEEM (pulsed EPR) spectroscopies. The three-pulse ESEEM spectrum of the superoxidized Mn(III)Mn(IV) enzyme obtained at 3429 G shows a frequency pattern with peaks at 0.60, 1.45, 2.06, and 5.03 MHz that is assigned to the magnetic coupling in the exact cancellation regime of one 14N atom that coordinates the Mn dimer, with magnetic parameters e2Qq = 2.34 MHz, eta = 0.51, and Aiso = 2.45 MHz. When the enzyme is chemically modified by reductive methylation, dramatic effects are detected both in the CW-EPR spectrum and in the ESEEM data. Spectral simulations of the CW-EPR signal suggest that the alterations in the spectra are related to the properties of the hyperfine coupling tensors of the Mn ions and of the g tensor, which changes from axial symmetry (gparallel - gperpendicular = 0.018) in the untreated catalase to a nearly isotropic symmetry (gparallel - gperpendicular = 0.002) in the modified enzyme. The three-pulse ESEEM spectrum of the catalase is also completely altered after the reductive methylation, with a rather different frequency pattern at 1.57, 2.35, 3.88, and 6.00 MHz. These data are interpreted as indicating that the hyperfine interaction from the coupled 14N donor is profoundly modified by the methylation treatment, changing from Aiso = 2.45 MHz to a larger value. The spectra are compared with ESEEM data obtained on two polynuclear Mn systems with 14N donors: the Mn cluster of Photosystem II inhibited by 14NH4Cl, and the model compound [Mn2(bipy)4(mu-O)2](ClO4)3. It is found that the ESEEM data measured on the untreated Mn(III)Mn(IV) catalase resemble those on the Photosystem II manganese site, suggesting that the coupled 14N coordinates the Mn dimer in an analogous fashion. By analogy to the mode of binding of ammonia in Photosystem II proposed by Britt et al. [Britt, R. D., Zimmermann, J. L., Sauer, K., & Klein, M. P. (1989) J. Am. Chem. Soc. 111, 3522-3532], it is proposed that a 14N atom bridges the two Mn ions in Mn(III)Mn(IV) catalase. By contrast, comparison of the data obtained on the methylated enzyme with those on the model compound suggests that the 14N couplings are similar in both systems; this is indicative of a terminal 14N ligand in the modified catalase.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunogenicity of biosynthetic human LysPro insulin compared to native-sequence human and purified porcine insulins in rhesus monkeys immunized over a 6-week period.

Development of insulin antibodies in rhesus monkeys was investigated after immunization with 3 forms of insulin in Freund's adjuvant. Insulins examined included: 1. biosynthetic LysPro insulin (LY275585), a new human insulin analog, 2. biosynthetic native-sequence human insulin, and 3. purified porcine insulin. Male monkeys, 4/insulin type, were immunized weekly over a 6-week period with increasing doses of insulin, ranging from 10 to 100 micrograms/monkey. An ELISA assay was used to measure IgG insulin antibodies in sera collected prior to immunization and 5, 10, and 16 days after final immunization. One monkey had detectable pretreatment levels of antibody. This monkey, which had been assigned to the LysPro insulin treatment group, responded to immunization with a peak antibody level of 20 micrograms/ml. IgG insulin antibody responses were not detected in any of the other monkeys. A passive cutaneous anaphylaxis (PCA) assay was used to measure IgE insulin antibodies in sera collected prior to immunization and 10 days after final immunization. No IgE antibodies were detected in any of the monkeys pre- or post-immunization. Considering that 1. an immunological adjuvant was used, 2. eleven of twelve monkeys failed to develop an antibody response, and 3. the IgG insulin antibody level observed in the single responding monkey was low, it was concluded that these insulins have an extremely weak immunogenic potential in rhesus monkeys. It is suggested that immunization of non-human primates with new therapeutic proteins in adjuvant may be a useful primary screen to determine their immunogenic potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of immunoreactivity of normal tissues from dogs, using monoclonal antibody B72.3.

Monoclonal antibody (MAB) B72.3, which recognizes human tumor-associated glycoprotein-72, has immunoreactivity for malignant epithelial neoplasms in human beings and dogs. To further characterize the range of immunoreactivity of MAB B72.3 in canine tissues, MAB B72.3 and 2 other tumor-associated glycoprotein-72 antibodies (MAB CC49 and CC83) were tested against a wide spectrum of normal tissues from dogs. Immunoreactivity was detected, using an avidin-biotin-complex immunoperoxidase method. Monoclonal antibody B72.3 did not stain most types of normal canine tissues, but various types of epithelial cells within the gastrointestinal and respiratory tract mucosae, salivary gland, esophagus, epididymis, uterus, thymus, hair follicle, and apocrine glands of the anal sac had variable staining with MAB B72.3. A similar range of immunoreactivity in comparable types of normal tissues was seen for MAB CC49 and CC83; however, MAB CC49, but not MAB B72.3 and CC83, stained the endothelium of capillaries and small vessels in most normal tissues. Staining of frozen and paraffin-embedded tissues was similar. In conclusion, we found that MAB B72.3, CC49, and CC83 had selected immunoreactivity for specific types of normal canine epithelial cells, especially those involved with mucin production.

Angular orientation of the stable tyrosyl radical within photosystem II by high-field 245-GHz electron paramagnetic resonance.

The 4 K 245-GHz/8.7-T electron paramagnetic resonance spectrum of the stable tyrosyl radical in photosystem II, known as TyrD., has been measured. Illumination at 200 K enhances the signal intensity of TyrD. by a factor of > 40 compared to the signal obtained from dark-adapted samples. This signal enhancement and the unusual line shape of the TyrD. resonance result from the magnetic dipolar coupling of the radical to the manganese cluster involved in oxygen evolution. The relative angular orientation of the manganese cluster with respect to TyrD. has been determined from line-shape analysis. The resonance arising from TyrD. in Tris-washed manganese-free photosystem II sample is also distorted. This effect probably originates from the influence of the nonheme iron on the spin relaxation of the tyrosyl radical. The relative angular orientation of the nonheme iron has also been determined. Oriented samples were used to determine the angular orientation of TyrD. with respect to the membrane plane. Combining angular data with published distances, we have constructed a three-dimensional picture of the relative positions of TyrD., the manganese cluster, and the nonheme iron. The data suggest a more symmetrical placement of the manganese relative to TyrD. and TyrZ, the tyrosine involved in electron transfer, than is usually assumed in current models of photosystem II.