The antimicrobial properties of magnesium monoperoxyphthalate hexahydrate

The antimicrobial properties of magnesium monoperoxyphthalate hexahydrate (MMPP), which is a stable, water-soluble solid peroxyacid, have been investigated. A 2% (w/w) solution of MMPP rapidly kills yeasts and vegetative bacteria and slowly inactivates bacterial endospores at 22°C. Biocidal activity, which is greater under mildly acidic conditions, is retained in the presence of organic contamination or hard water. Sporicidal action is greatly increased by a moderate rise in temperature or by using MMPP in combination with propan-2-ol. Such mixtures appear to be suitable for use as liquid chemical sterilants.     

Studies of the in vitro oxidation of 1-methyl-4-(1-methylpyrrol-2-yl)-4-piperidinol and its dehydration product 1,2,3,6-tetrahydro-1-methyl-4-(methylpyrrol-2-yl) pyridine by human monoamine oxidases A and B

The ability of highly purified preparations of human monoamine oxidase A and B (MAO A and B) to utilize 1-methyl-4-(1-methylpyrrol-2-yl)-4-piperidinol (MMPP) and its dehydration product 1,2,3,6-tetrahydro-1-methyl-4-(methylpyrrol-2-yl) pyridine (TMMP) as substrates was investigated. The results showed that TMMP was a substrate for both forms of MAO with Km,app values of approximately 60 microM. However, MAO B had a Vmax,app for TMMP about 30-fold greater than MAO A. Additional studies revealed that MMPP was a poor substrate of only MAO B (Km,app = 9.5 mM) and that acid treatment of MMPP led to the formation of a product that could be readily oxidized by both MAO A and B. Similar acid pretreatment of TMMP yielded a product that was a much poorer substrate for MAO B than the parent compound. These results may partially explain why orally administered MMPP produces neurotoxicity in monkeys and TMMP fails to induce chemical parkinsonism.     

Magnesium monoperoxophtalate: an efficient single oxygen atom donor in DNA cleavage catalyzed by metalloporphyrin

The peracid compound magnesium monoperoxophtalate (MMPP), water-soluble at physiological pH, behaves as a promising oxygen donor in the oxidative cleavage of DNA catalyzed by meso-tetrakis-(4-N-methylpyridyl)porphyrinatomanganese(III) pentaacetate. Shown on cleavage of supercoiled phi X174 DNA, MMPP is significantly more efficient than KHSO5, another recently developed single oxygen atom donor (Fouquet et al., J. Chem. Soc., Chem. Commun., 1987, 1169).     

Oxidative cleavage of DNA mediated by hybrid metalloporphyrin-ellipticine molecules and functionalized metalloporphyrin precursors

The nuclease activity of functionalized metalloporphyrins 1-8 and hybrid metalloporphyrin-ellipticine molecules 10-16 in the presence of potassium monopersulfate (KHSO5) or magnesium monoperoxyphthalate (MMPP), water-soluble oxygen atom donors at physiological pH, toward double-stranded phi X174 DNA is reported. The DNA cleavage efficiency as a function of the nature of functionalized metalloporphyrins, the length of the linkage between the two parts of the hybrid molecule, viz., metalloporphyrin and 9-methoxyellipticine, the nature of the central metal atom (Mn, Fe, or Zn) the ionic strength, and the nature of the oxygen donor has been studied. Single-strand breaks (SSBs) are observed on double-stranded DNA with a short incubation time of 2 min in the presence of manganese derivatives of both metalloporphyrins and hybrid molecules. Owing to their cytotoxic and nuclease activity, these new water-soluble hybrid molecules may be considered as efficient bleomycin models based on cationic metalloporphyrins.     

Site-specific cross-linking of proteins through tyrosine hexahistidine tags

The genetic addition of hexahistidine (H(6)) tags is widely used to isolate recombinant proteins by immobilized metal-affinity chromatography (IMAC). Addition of a tyrosine residue to H(6) tags enabled proteins to be covalently cross-linked under mild conditions in a manner similar to the natural, site-specific cross-linking of tyrosines into dityrosine. A series of seven hexahistidine tags with tyrosines placed in various positions (H(6)Y tags) were added to the amino terminus of the I28 immunoglobulin domain of the human cardiac titin. The H(6)Y-tagged I28 dimerized in the presence of excess Ni(2+) with a K(D) of 200 microM. Treatment of Ni(2+)-dimerized H(6)Y-I28 with an oxidant, monoperoxyphthalic acid (MMPP) or sodium sulfite, resulted in covalent protein multimerization through chelated Ni(2+)-catalyzed cross-linking of the Y residues engineered into the H(6) tag. The protein oligomerization was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The presence of dityrosine in the cross-linked proteins was confirmed by fluorescence emission at 410 nm. Proteins lacking the Y residue in the H(6) tag treated with the same oxidative conditions did not cross-link or exhibit dityrosine fluorescence, despite the presence of an endogenous Y residue. The method may have potential uses in other protein conjugation applications such as protein labeling and interfacial immobilization of proteins on artificial surfaces.     

King''s Formula for the Mutation Load with Epistasis

A formula by J. L. King gives the equilibrium mutation load as L = 2 sigma ui(1 - qi)/z - x) in which ui is the mutation rate to deleterious alleles at the ith locus, qi is the frequency of mutant alleles at this locus, x is the mean number of such mutant genes per individual before selection, z is the mean number in individuals eliminated by selection, and the summation is over all relevant loci. We show that this rule is inaccurate for intense selection and that a correct formula is L = 2 sigma ui(1 - qi) w/(z - x) = 2U w/(z - x) = 2U/(z - x + 2U) in which U is the mean number of new mutations per haploid genome in the population and w is the mean relative fitness before selection. If w/(z - x) less than 1/2, the mutation load is less than the Haldane value (U less than or equal to L less than or equal to 2U) and can be considerably less. In a diploid asexual population, however, with independent occurrence of mutations, L = 1 - e-2U regardless of the mode of selection.     

Effect of calcium on the morphology and functionality of whey protein nanofibrils

Self-assembly of amyloid-like nanofibrils during heating of bovine whey proteins at 80 °C and pH 2 is accelerated by the presence of NaCl and/or CaCl(2), but the rheological consequences of accelerated self-assembly are largely unknown. This investigation focused on the impact of CaCl(2) on the evolution of rheological properties and fibril morphology of heated whey protein isolate (WPI), both during self-assembly at high temperature and after cooling. Continuous rotational rheometry of heated 2% w/w WPI showed a nonlinear effect of CaCl(2) on the viscosity of fibril dispersions, which we attributed to effects on fibril flexibility and thus the balance between intrafibril and interfibril entanglements. Small-amplitude oscillatory measurements made in situ during heating of 10% w/w WPI at 80 °C suggest that CaCl(2) is not involved in either fibril structure or gel structure, and this was confirmed with dialysis experiments.     

Reduction of 2-acetylaminofluorene-induced unscheduled DNA synthesis in human and rat hepatocytes by butylated hydroxytoluene

Butylated hydroxytoluene (BHT) protected against DNA damage induced in rat hepatocytes by 2-acetylaminofluorene (2AAF) or N-hydroxy 2AAF as shown by a marked reduction of unscheduled DNA synthesis. BHT also inhibited 2AAF-induced DNA damage (as shown by reduced repair) in human hepatocytes. In addition, rats pre-treated with BHT in the diet (0.5% w/w for 10 days) provided hepatocytes which exhibited less unscheduled DNA synthesis than did hepatocytes from control rats when these cells were exposed to either 2AAF or N-hydroxy 2AAF. The results indicate both direct (in vitro) and indirect (by pre-treatment in vivo) inhibitory effects of BHT on the genotoxicity of 2AAF in liver cells, in accord with the reported anti-tumorigenicity in the liver. This effect contracts with a BHT-mediated increase in the efflux of 2AAF-derived mutagens from liver cells which may contribute to enhanced extrahepatic carcinogenesis.     

Importance of hydration for gramicidin-induced hexagonal HII phase formation in dioleoylphosphatidylcholine model membranes

The macroscopic organization, lipid head group conformation, and structural and dynamic properties of 2H2O were investigated in dioleoylphosphatidylcholine (DOPC) model systems of varying gramicidin and 2H2O (or H2O) content by means of small-angle X-ray diffraction and 31P and 2H NMR. At low stages of hydration, N less than 6 (N = 2H2O/DOPC molar ratio), a single lamellar phase is observed in which the gramicidin molecules become preferentially hydrated upon increasing N. For 6 less than N less than 12 phase separation occurs between a gramicidin-poor and a gramicidin-rich lamellar phase. This latter phase is characterized by a smaller repeat distance and decreased DOPC head group order. For N greater than 12, the gramicidin-rich lamellar phase converts to a hexagonal HII phase. Thus, hydration of gramicidin is a prerequisite for HII phase formation in the DOPC/gramicidin system. The HII phase is very rich in gramicidin and 2H2O (gramicidin:DOPC:H2O = 1:1.1:0.9 w/w/w). A model is proposed in which self-assembly of hydrated gramicidin molecules into domains of a specific structure plays a determinant role in the formation of the HII phase by gramicidin.     

Hepatic oxidative stress and related defenses during treatment of mice with acetylsalicylic acid and other peroxisome proliferators

The peroxisome proliferators perfluorooctanoic acid (PFOA; 0.02% w/w), perfluorodecanoic acid (PFDA; 0.02%, w/w), nafenopin (0.125%, w/w), clofibrate (0.5%, w/w), and acetylsalicylic acid (ASA; 1%, w/w) were administered to male C57 BL/6 mice in their diet for two weeks. Parameters for Fe³⁺ ADP, NADPH or ascorbic acid-initiated lipid peroxidation in vitro were measured. Approximately a twofold increase in susceptibility to lipid peroxidation was obtained for all the peroxisome proliferators tested. Cotreatment of mice with the peroxisome proliferator ASA (1%, w/w) and a catalase inhibitor, 3-amino-1,2,4-triazole (AT; 0.4%, w/w) for 7 days resulted in little inhibition of peroxisome proliferation, an elevated level of H₂O₂ in vivo, and total inhibition of the increased susceptibility to lipid peroxidation in vitro. No increase in lipid peroxidation in vivo was observed. Certain antioxidant enzymes (DT-diaphorase, superoxide dismutase, glutathione transferase, glutathione peroxidase, and glutathione reductase) and components (ubiquinone and α-tocopherol) were also measured. The results showed that there was some induction of these antioxidant enzymes and components by ASA or aminotriazole, except for glutathione peroxidase and superoxide dismutase, which were inhibited. The possible involvement of oxidative stress in the carcinogenicity of peroxisome proliferators is discussed.     

Role of cardiac eNOS expression during pregnancy in the coupling of myocardial oxygen consumption to cardiac work

We assessed whether pregnancy results in enhanced nitric oxide (NO)-mediated control of myocardial oxygen consumption. Rats were studied before (C), at 1 wk (1w) or 2 wk (2w) of pregnancy, and at 4 days after giving birth (-4d). Left ventricular endothelial NO synthase (eNOS) protein expression was determined by immunoblotting. Oxygen consumption of left ventricular tissue samples was measured in vitro in response to increasing doses of bradykinin with or without addition of the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Echocardiography indicated an increased cardiac output during pregnancy. Myocardial eNOS protein expression significantly increased by 46 +/- 9 and 39 +/- 8% at 1w and 2w, respectively, and returned to control levels at -4d. Bradykinin (10(-4) M) decreased cardiac oxygen consumption in a NO-dependent manner by 17 +/- 2% at C, by 21 +/- 2% at 1w, by 24 +/- 2% at 2w (P < 0.05 vs. C and -4d), and by 18 +/- 1% at -4d. Myocardial eNOS protein expression is transiently increased during pregnancy in rats, and this increase is associated with enhanced NO-dependent control of myocardial oxygen consumption at a time when cardiac output is increased.     

Evaluation of Hill slopes and Hill coefficients when the saturation binding or velocity is not known.

1. The Hill coefficient (nH), an often-used measure of deviations from hyperbolic behaviour (nonhyperbolicity) in kinetic and binding systems, is usually estimated from the maximum or minimum slope of the Hill plot. The method depends strongly on the assumed magnitude of the asymptotic velocity (V) or binding (P) whose evaluation may be difficult in nonlinear/co-operative systems. Therefore, alternative procedures were devised for the estimation nH which do not require the prior knowledge of V or P. 2. When pairs of velocity/binding readings (v and w) are obtained at concentrations of c and alpha c, respectively (where alpha is a fixed constant), then the relation between w and v is described by a hyperbola, provided that Hill's equation is valid. In this case, linearizing plots, v/w versus v, w versus, w/v, and 1/w versus 1/v, can be used for estimation of the degree of the equation. However, if the Hill expression is applicable, these methods are not efficient and traditional procedures, particularly nonlinear regression, should be used. 3. The 'linearizing' plots of the Hill equation can be applied advantageously for the evaluation of the Hill slope and of nH also in the general case, when the Hill expression is actually not valid, provided that deviations from hyperbolic behaviour are positive. Appropriately extrapolated intercepts of the first two plots estimate alphanH. Furthermore, the slope of the third plot yields, similarly to the method of Kurganov et al., a continuous measure of the Hill slope (including its maximum) at all concentrations. The agreement is, at positive nonhyperbolicities, excellent theoretical values of Hill slopes and coefficients and those estimated by the proposed methods. 4. A coefficient of nonhyperbolicity (theta) is defined for 2nd-degree rate equations which provides a quantitative measure of positive or negative deviation from first-degree, hyperbolic characteristics. It is closely related to the Hill coefficient.     

Pretreatment of milk thistle seed to increase the silymarin yield: an alternative to petroleum ether defatting

Milk thistle (Silybum marianum L.) seed meal is extracted for the flavonolignans, silychristin, silydianin, silybinin A, silybinin B, isosilybinin A and isosilybinin B, which are collectively known as the silymarin complex. To obtain the flavonolignans, the meal is usually treated with successive washes of petroleum ether to remove the lipids, followed by extraction of the flavonolignans with ethanol. This work examines the possible replacement of petroleum ether and ethanol by water or other aqueous solutions in these processes. To replace petroleum ether, pretreatments with 1.2% NaOH (w/w), 1.5% H2SO4 (w/w), 2% NaHCO3 (w/w), 0.14% cellulase and water were investigated. Of these pretreatments, 1.5% H2SO4 and water produced similar flavonolignan yields as petroleum ether. Results established that pretreating the milk thistle seed meal with 1.5% H2SO4 (w/w) at 50 degrees C for 18 h could replace the petroleum ether pretreatment. In addition, it was shown that similar amounts of flavonolignan could be recovered with a 1.5% H2SO4/water (100 degrees C) extraction as with a petroleum ether/ethanol extraction. Although cellulase pretreatment was not examined extensively, significant advances in cellulase effectiveness and cost have occurred in the past few years by companies such as Genencor International and Novozymes. These advances should help to make enzyme use for cellulose conversion, as well as extraction pretreatment, technically and economically feasible.     

Purification and Characterization of the Egg Jelly Macromolecules, Sialoglycoprotein and Fucose Sulfate Glycoconjugate, of the Sea Urchin Hemicentrotus Pulcherrimus

A sialoglycoprotein and a fucose sulfate glycoconjugate (FSG) were purified from egg jelly of the sea urchin Hemicentrotus pulcherrimus . Sialoglycoprotein which consisted of sialic acid (90%, w/w) and protein (10%, w/w) did not cause induction of the acrosome reaction and sperm isoagglutination. FSG which contained one mol sulfate/mol fucose possessed 2.0 times protein to fucose by weight. The proteins in intact FSG were separated to two major (258 kDa and 237 kDa) and one minor (120 kDa) proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol (2-ME) while the proteins could not be separated by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. However, after carboxymethylation of FSG, two major (260 kDa and 240 kDa) proteins and two minor (140 kDa and 135 kDa) proteins were separated from the fucose sulfate moiety by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. When FSG was first carboxymethylated with non-radioactive iodoacetic acid and then reduced with 2-ME and finally carboxymethylated with ¹⁴C-iodoacetic acid, the most of radioactivity was detected in 140 kDa and 135 kDa proteins. Carboxymethylted-FSG was less potent than intact FSG in induction of the acrosome reaction. Fucoidan, a fucose sulfate polymer, did not induce the acrosome reaction.     

The Saccharomyces cerevisiae orthologue of the human protein phosphatase 4 core regulatory subunit R2 confers resistance to the anticancer drug cisplatin

The anticancer agents cisplatin and oxaliplatin are widely used in the treatment of human neoplasias. A genome-wide screen in Saccharomyces cerevisiae previously identified PPH3 and PSY2 among the top 20 genes conferring resistance to these anticancer agents. The mammalian orthologue of Pph3p is the protein serine/threonine phosphatase Ppp4c, which is found in high molecular mass complexes bound to a regulatory subunit R2. We show here that the putative S. cerevisiae orthologue of R2, which is encoded by ORF YBL046w, binds to Pph3p and exhibits the same unusually high asymmetry as mammalian R2. Despite the essential function of Ppp4c-R2 in microtubule-related processes at centrosomes in higher eukaryotes, S. cerevisiae diploid strains with homozygous deletion of YBL046w and two or one functional copies of the TUB2 gene were viable and no more sensitive to microtubule-depolymerizing drugs than the control strain. The protein encoded by YBL046w exhibited a predominantly nuclear localization. These studies suggest that the centrosomal function of Ppp4c-R2 is not required or may be performed by a different phosphatase in yeast. Homozygous diploid deletion strains of S. cerevisiae, pph3Delta, ybl046wDelta and psy2Delta, were all more sensitive to cisplatin than the control strain. The YBL046w gene therefore confers resistance to cisplatin and was termed PSY4 (platinum sensitivity 4). Ppp4c, R2 and the putative orthologue of Psy2p (termed R3) are shown here to form a complex in Drosophila melanogaster and mammalian cells. By comparison with the yeast system, this complex may confer resistance to cisplatin in higher eukaryotes.     

Cryoprotection of red blood cells by a 2,3-butanediol containing mainly the levo and dextro isomers.

A 2,3-butanediol containing 96.7% (w/w) racemic mixture of the levo and dextro isomers and only 3.1% (w/w) of the meso isomer (called 2,3-butanediol 97% dl) has been used for the cryoprotection of red blood cells. The erythrocytes were cooled to -196 degrees C at rates between 2 and 3500 degrees C/min, followed by slow or rapid warming. Up to 20% (w/w) of this polyalcohol, only the classical peak of survival is observed, as with up to 20% (w/w) 1,2-propanediol or 1,3-butanediol. Twenty percent 2,3-butanediol 97% dl can protect red blood cells very efficiently. The maximum survival, of 90%, as with 20% glycerol, is a little lower than with 20% 1,2-propanediol and higher than with 20% 1,3-butanediol. Fifteen percent 2,3-butanediol protects fewer red blood cells than 15% glycerol or 1,2-propanediol, with a maximum survival of about 80%. The best cryoprotection by 30% 2,3-butanediol 97% dl is obtained at the slowest cooling and warming rates, where survival approaches 90%. After a minimum, an increase of survival is observed at the fastest cooling rates, which would correspond to complete vitrification. These rates are lower than with 30%, 1,2-propanediol or 1,3-butanediol, in agreement with the higher glass-forming tendency of 2,3-butanediol 97% dl solutions. In agreement with the remarkable physical properties of its aqueous solutions, the present experiments also suggest that 2,3-butanediol containing mainly the levo and dextro isomers could be a very useful cryoprotectant for organ cryopreservation. However, it would perhaps be better to use it in combination with other cryoprotectants, since it is a little more toxic than glycerol or 1,2-propanediol at high concentrations.     

Postnatal ovary is necessary for the maturation of estradiol-induced luteinizing hormone and prolactin surges in female rats

The role of postnatal ovary in the maturation of estradiol (E2)-induced luteinizing hormone (LH) and prolactin (PRL) surges was examined in female rats of Wistar-Imamichi strain. Animals were bilaterally ovariectomized at 24 h after birth, 1 week (w), 2 w, 3 w, 4 w or 6 w of age. At about 10 w of age, every group was primed with estradiol benzoate (E2B) for two days, and on the third day was decapitated at either 0900 h or 1900 h. Anterior pituitary (AP) LH and PRL content was determined in every group of no E2B treatment. Surge-like secretions of LH and PRL were observed at 1900 h, only in rats ovariectomized on or after 4 w of age. AP LH and PRL content was the higher, as ovariectomy was delayed. These results indicate that postnatal ovary is necessary for the maturation of E2-induced LH and PRL surges. Such an effect of ovary is mediated at least by its stimulation of AP LH and PRL content.