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.

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.

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.

The g = 2 multiline EPR signal of the S2 state of the photosynthetic oxygen-evolving complex originates from a ground spin state.

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.

Factors influencing the formation of modified S2 EPR signal and the S3 EPR signal in Ca(2+)-depleted photosystem II.

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.

Monoclonal antibody L/1C2 reactive with a human carcinoma associated antigen.

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.

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.

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.

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.

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.

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)

Target organ toxicity and leukopenia in Fischer 344 rats given intravenous doses of vinblastine and desacetyl vinblastine.

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.

The manganese center of oxygen-evolving and Ca(2+)-depleted photosystem II: a pulsed EPR spectroscopy study.

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.

EPR signals from modified charge accumulation states of the oxygen evolving enzyme in Ca2+-deficient photosystem II.

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.

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.

Mycotoxicosis produced in swine by cultural products of an isolate of Aspergillus ochraceus. I. Clinical observations and pathology.

Pigs fed a ration, 25% of which was rice culture, of Aspergillus ochraceus lost weight or failed to gain and became depressed. Some pigs died and most developed subcutaneous edema, hydrothorax, hydroperitoneum, pulmonary atelectasis, edema of the mesentery and perirenal edema. Microscopic lesions in addition to edema were primarily renal and consisted of tubular degeneration and necrosis, hyaline tubular casts, interstitial fibrosis and tubular cell regeneration. The first change found after 3 days was cytoplasmic vacuolation of the convoluted and straight segments of the proximal tubules. Necrotic proximal tubules were found after 4 days and after 9 days degeneration and necrosis involved predominantly proximal tubular segments. Pigs fed a ration, 12.5% of which was rice culture, for 8 weeks did not develop perirenal edema but had firm kidneys. Extensive interstitial fibrosis of the cortical labyrinth was the principal change. Within the fibrous connective tissue, some tubules were necrotic and others were atrophied.

Mycotoxicosis produced in swine by cultural products of an isolate of Aspergillus ochraceus. II. Clinicopathologic changes.

Rice culture of a toxigenic strain of Aspergillus ochraceus was fed at a concentration of 25% to weanling pigs for 10 days. The clinicopathological abnormalities reflected renal damage. Activities of lactic dehydrogenase, glutamic-oxaloacetic transaminase and isocitrate dehydrogenase were increased in the urine but not in the serum. Serum concentrations of urea nitrogen and creatinine were high. Cellular and granular casts, blood, protein, and glucose were in the urine of pigs fed toxic diet. Serum concentrations of K+, Na+ and Cl- were unchanged, but concentrations of these electrolytes were reduced in the urine.

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.