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Following surgery of cholesteatoma, a patient developed a chronic infection of the external auditory canal, including extended-spectrum β-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.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

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 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.

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.

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.

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.

[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.

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.

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)

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)

The environment of the multi-manganese center in the O2-evolving complex (OEC) of plant photosystem II (PS II) under conditions of Ca2+ depletion has been probed using pulsed electron paramagnetic resonance (EPR) spectroscopy, and the following results are reported: (1) In Ca(2+)-depleted PS II membranes treated with the chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), the modified Mn EPR signal arising from the OEC in the S2 state and the split EPR signal from the S3 state could be detected in the absorption mode by recording the amplitude of a two-pulse echo as a function of the external magnetic field. The formation of the S3 signal (g approximately 2.004; delta Hpp = 164 G) is not accompanied by the disappearance of the Mn EPR signal, although the signal becomes difficult to detect in CW EPR. This result supports the previous interpretation of the split S3 EPR signal as arising from the interaction of an organic radical with the Mn cluster [Boussac, A., Zimmermann, J. L., Rutherford, A. W., & Lavergne, J. (1990) Nature 347, 303-306]. (2) The two-pulse electron spin echo envelope modulation (ESEEM) spectra of the S2 state formed in Ca(2+)-depleted PS II membranes obtained from 14N- and 15N-labeled material are different. This indicates that nitrogen nuclei from nitrogen-containing protein residues are coupled to the Mn center in the S2 state of the inhibited enzyme. In addition, comparison with the two-pulse ESEEM data obtained for the S2 state in the untreated enzyme suggests that the coupling may be altered by the Ca2+ depletion and/or EGTA treatment. (3) The treatment of Ca(2+)-depleted PS II membranes with sodium pyrophosphate also induced a stable S2 state characterized by a modified multiline EPR signal that is similar to that obtained in EGTA-treated PS II membranes. Comparison of the ESEEM data obtained for the pyrophosphate and 14N and 15N samples treated with EGTA suggests that the modification induced by the EGTA treatment is accompanied by the binding of (an) EGTA molecule(s) to or near the Mn center. (4) ESEEM data obtained for the S3 state formed in the pyrophosphate or EGTA-treated enzyme are quite similar to those obtained for the corresponding S2 state. The data are also compared with ESEEM data obtained on oxidized 4(5)-methylimidazole obtained by UV irradiation. These results are discussed with respect to the current assignment of the S3 radical as arising from oxidation of a histidine residue.

The amplitude of the g = 2 Mn 'multiline' EPR signal of the S2 state of the photosynthetic oxygen-evolving complex varies inversely with temperature, indicating that this signal arises from a ground spin state. Electron spin echo experiments at temperatures of 4.2 K and 1.4 K show such Curie-law behavior of the g = 2 multiline EPR signal, as do continuous-wave EPR experiments performed at a non-saturating microwave power in the range from 15.0 K to 4.2 K.

The Oxygen evolving complex of plant photosystem II is made of a manganese cluster that gives rise to a low temperature EPR multiline signal in the S(2) oxidation state. The origin of this EPR signal has been addressed with respect to the question of the magnetic couplings between the electron and nuclear spins of the four possible Mn ions that make up this complex. Considering Mn(III) and Mn(IV) as the only possible oxidation states present in the S(2) state, and no large anisotropy of the magnetic tensors, the breadths of the EPR spectra calculated for dimers and trimers with S = (1/2) have been compared with that of the biological site. It is concluded that neither a dinuclear nor a trinuclear complex made of Mn(III) and Mn(IV) can be responsible for the multiline signal; but that, by contrast, a tetranuclear Mn complex can be the origin of this signal. The general shape of the experimental spectrum, its particular hyperfine pattern, the positions of most of the hyperfine lines and their relative intensities can be fit by a tetramer model described by the following six fitting parameters: g approximately 1.987, A(1) approximately 122.4 10(-4) cm(-1), A(2) approximately 87.2 10(-4) cm(-1), A(3) approximately 81.6 10(-4) cm(-1), A(4) approximately 19.1 10(-4) cm(-1) and deltaH = 24.5 G. A second model described by parameters very close to those given above except for A(4) approximately 77.5 10(-4) cm(-1) gives an equally good fit. However, no other set of parameters gives an EPR spectrum that reproduces the hyperfine pattern of the S(2) multiline signal. This demonstrates that in the S(2) state of the oxygen evolving complex, the four manganese ions are organized in a magnetic tetramer.

The toxicity and leukopenia produced by vinblastine or desacetyl vinblastine were established in 1-month studies in rats. Groups of male Fischer 344 rats were given weekly intravenous doses of vinblastine or desacetyl vinblastine at doses of 0.08, 0.16, 0.32, 0.64, or 1.28 mg/kg. The target organ toxicity was similar for both compounds. Decreased cell production in the thymus, testes, and bone marrow was produced in the animals of the two highest dose groups for both compounds with the degree of severity greater in the highest dose group. All high dose rats (1.28 mg/kg) given desacetyl vinblastine and three rats given vinblastine died prior to study termination. Body weight loss was more pronounced in high dose rats given desacetyl vinblastine, but at lower doses there were no significant differences in body weight reduction for rats receiving either compound. Leukopenia occurred at all dose levels of 0.32 mg/kg and higher. During the first 2 weeks of the study, rats given 1.28 mg/kg of desacetyl vinblastine had a greater leukopenic response than rats given 1.28 mg/kg of vinblastine. It is concluded that both compounds produced similar target organ toxicity and leukopenia without deaths at doses of 0.32 and 0.64 mg/kg given once a week for 4 weeks. At the high dose (1.28 mg/kg) desacetyl vinblastine was more toxic than vinblastine resulting in greater mortality and body weight reduction.

NaCl/EGTA-washing of photosystem II (PS-II) results in the removal of Ca2+ and the inhibition of oxygen evolution. Two new EPR signals were observed in such samples: a stable and modified S2 multiline signal and an S3 signal [(1989) Biochemistry 28, 8984-8989]. Here, we report what factors are responsible for the modifications of the S2 signal and the observation of the S3 signal. The following results were obtained. (i) The stable, modified, S2 multiline signal can be induced by the addition of high concentrations of EGTA or citrate to PS-II membranes which are already inhibited by Ca(2+)-depletion. (ii) The carboxylic acids act in the S3-state, are much less effective in S2 and have no effect in the S1-state. (iii) The extrinsic polypeptides (17- and 23-kDa) are not required to observe either the modified S2 signal or the S3 signal. However, they do influence the splitting and the lifetime of the S3 signal, and they seem to have a slight influence on the hyperfine pattern of the S2 signal. (iv) The S3 signal can be observed in Ca(2+)-depleted PS-II which does not exhibit the modified multiline signal. Then, it is proposed that formation of histidine radical during the S2 to S3 transition in Ca(2+)-depleted PS-II [(1990) Nature 347, 303-306] also occurs in functional PS-II.

A murine IgG3 monoclonal antibody which defines a human carcinoma associated antigen is described. The antibody, designated L/1C2, was made against a human lung squamous cell carcinoma line designated USCLS-1. It reacts with the surface of 15 of 16 viable human carcinoma cell lines and was detected in 70 of 78 frozen sections of human carcinomas. Melanoma cell lines and frozen sections of melanomas and a lymphoma were unreactive. Normal tissue reactivity included vessels, plus some ducts, glandular structures, and epithelial surfaces. Similar normal tissue reactivity patterns were seen with Rhesus monkey tissue samples. Immunoprecipitation studies indicate that L/1C2 reacts with a glycoprotein doublet which migrates in the range of 110,000 to 140,000 Mr under reducing conditions. Fluorescence analysis suggests this antigen is internalized following reaction with the L/1C2 antibody. Using in vitro human tumor cell growth inhibition assays, it was possible to achieve significant growth inhibition with L/1C2, while another target cell-reactive antibody in the same assay had no inhibitory effect.

Photosystem II enriched membranes were depleted of Ca2+ and the 17- and 23-kDa polypeptides by treatment with NaCl and EGTA. The 17- and 23-kDa polypeptides were then reconstituted. This preparation was incapable of O2 evolution until Ca2+ was added. An EPR study revealed the presence of two new EPR signals. One of these is a modified S2 multiline signal with an isotropic g value of 1.96 with at least 26 hyperfine peaks (average spacing 55 G) distributed over approximately 1600 G. The other is a near-Gaussian signal with an isotropic g value of 2.004, which is attributed to a formal S3 state. Experiments involving the interconversion of these signals and the effect of Ca2+ and Sr2+ rebinding provide evidence for these assignments. From these results the following conclusions are drawn: (1) These results are consistent with our earlier demonstration that charge accumulation is blocked after formation of S3 when Ca2+ is deficient. (2) Binding of the 17- and 23-kDa polypeptides to photosystem II in the absence of Ca2+ results in the perturbation of the Mn cluster. This is taken as a further indication that the Ca2+-binding site is close to or even an integral part of the Mn cluster. (3) The S3 signal may arise from an organic free radical interacting magnetically with the Mn cluster. However, other possible origins for this signal, including the Mn cluster itself, must also be considered.

An immunoconjugate (PF1/D-DAVLBHYD), made with the squamous carcinoma reactive monoclonal antibody PF1/D and a derivative of vinblastine, DAVLBHYD, was shown to suppress established T222 human tumor nude mouse xenografts using a multidose protocol. Treatments of xenograft-bearing mice with free drug, free antibody, or a mixture of the two, were unsuccessful at achieving suppression without associated toxicity, using otherwise identical protocols. A Vinca conjugate with a related squamous carcinoma reactive monoclonal antibody, PF1/B, was shown to have similar tumor suppressive activity. In a dual immunoconjugate therapy protocol, PF1/D-DAVLBHYD and PF1/B-DAVLBHYD had additive antitumor effects which were consistent with their complementary tumor reactivity.