Reactivity of Hg(II) with superoxide: Evidence for the catalytic dismutation of superoxide by HG(II)

Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O) and hydrogen peroxide (H₂O₂) production by rat kidney mitochondria, Hg(II) (1–6 μM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H₂O₂ production but an apparent decrease in mitochondrial O production. Hg(II) also inhibited O-dependent cytochrome c reduction (IC₅₀ ≈?2–3 μM) when O was produced from xanthine oxidase. In contrast, Hg(I) did not react with O in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O by Hg(II). Hg(II) also inhibited the reactions of KO₂ (i.e., O) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO₂ in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO₂. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O dismutation, leading to increased production of H₂O₂ by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.     

Estimation of Trichoderma QM 9414 biomass and growth rate by indirect means

Trichoderma QM 9414 was aerobically grown on glucose as the sole carbon and energy sources in a chemostat culture. The specific rates of glucose consumption (Q_G), oxygen consumption (Q), and carbon dioxide production (Q) at the steady state were measured to estimate the growth and maintenance requirements. From the results it was estimated that 2 mol adenosine triphosphate (ATP) were produced when1 mol NADH was oxidized through the respiratory chain of this microorganism. The true growth yield for ATP (Y_ATP) and specific ATP consumption rate for maintenance (Q) calculated with this value were 0.0106 g dry cell/mmol ATP and 5.2 mmol ATP/g dry cell/hr, respectively. Using the relationships between specific growth rate (μ) and (Q) and between μ and Q_G obtained from chemostat-culture data, cell and glucose concentration histories were estimated from the carbon dioxide production rate during the batch culture. The estimated cell concentrations agreed with the experimentally measured values. Glucose concentration were slightly overestimated.     

Levels of DNA strand breaks and superoxide in phorbol ester-treated human granulocytes

Phorbol ester treatment of granulocytes triggers release of superoxide (O) and a concomitant burst of DNA strand breaks. The relationship between the amount of O and the number of DNA breaks has not previously been explored. To quantify the relatively large amount of O generated over a 40-min period by 1 × 10⁶ granulocytes/mL, a discontinuous “10-min pulse” method employing cytochrome c was used; 140 nmol O per 1 × 10⁶ cells was detected. DNA strand breaks were quantified by fluorimetric analysis of DNA unwinding (FADU). To vary the level of O released by cells, inhibitors of the respiratory burst were used. Sodium fluoride (1–10 mM) and staurosporine (2–10 nM) both inhibited O production. In both cases, however, inhibition of strand breakage was considerably more pronounced than inhibition of O. Zinc chloride (50–200 μM) inhibited both O and DNA breaks, approximately equally. Dinophysistoxin-1 (okadaic acid) inhibited O production more effectively than it inhibited DNA breaks. O dismutes to H₂O₂, a reactive oxygen species known to cause DNA breaks. The addition of catalase to remove extracellular H₂O₂ had no effect on DNA breakage. Using pulse field gel electrophoresis, few double-stranded breaks were detected compared to the number detected by FADU, indicating that about 95% of breaks were single-stranded. The level of DNA breaks is not directly related to the amount of extracellular O or H₂O₂ in PMA-stimulated granulocytes. We conclude that either an intracellular pool of these reactive oxygen species is involved in breakage or that the metabolic inhibitors are affecting a novel strand break pathway. J. Cell. Biochem. 66:219–228, 1997. © 1997 Wiley-Liss Inc.     

Superior exercise performance in lifelong Tibetan residents of 4,400 m compared with Tibetan residents of 3,658 m

Few environments challenge human populations more than high altitude, since the accompanying low oxygen pressures (hypoxia) are pervasive and impervious to cultural modification. Work capacity is an important factor in a population's ability to thrive in such an environment. The performance of work or exercise is a measure of the integrated functioning of the O₂ transport system, with maximal O₂ uptake (VO) a convenient index of that function. Hypoxia limits the ability to transport oxygen: maximal O₂ uptake decreases with ascent to high altitude, and years of high altitude residence do not restore sea level VO values. Since Tibetans live and work at some of the highest altitudes in the world, their ability to exercise at very high altitude (<4,000 m) may define the limits of human adaptation to hypoxia. We transported 20 Tibetan lifelong residents of ≥4,400 m down to 3,658 m in order to compare them with 16 previously studied Tibetan residents of Lhasa (3,658 m). The two groups of Tibetans were matched for age, weight, and height. All studies were performed in Lhasa within 3 days of the 4,400 m Tibetans' arrival. Standard test protocol and criteria were used for attaining VO on a Monark bicycle ergometer, while measuring oxygen uptake (VO₂, ml/kg − min STPD), heart rate (bpm), minute ventilation (VE, 1/min BTPS), and arterial oxygen saturation (Sa, %). The 4,400 m compared with 3,658 m residents had, at maximal effort, similar VO₂ (48.5 ± 1.2 vs. 51.2 ± 1.4 ml/kg − min, P = NS), higher workload attained (211 ± 6 vs. 177 ± 7 watts, P < 0.01), lower heart rate (176 ± 2 vs. 191 ± 2 bpm, P < 0.01), lower ventilation (127 ± 5 vs. 149 ± 5 l/min BTPS, P < 0.01), and similar Sa(81.9 ± 1.0 vs. 83.7 ± 1.2%, P = NS). Furthermore, over the range of submaximal workloads, 4,400 m compared with 3,658 m Tibetans had lower VO₂ (P < 0.01), lower heart rates (P < 0.01), and lower ventilation (P < 0.01) and Sa (P < 0.05). We conclude that Tibetans living at 4,400 m compared with those residing at 3,658 m achieve greater work performance for a given VO₂ at submaximal and maximal workloads with less cardiorespiratory effort. Am J Phys Anthropol 105:21–31, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of changes in cytosolic Ca2+ and Na+ concentrations in rat submandibular gland acini exposed to carbachol and ATP

The relationship between cytosolic concentrations of Ca²⁺ (Ca) and Na⁺ (Na) were studied in preparations of rat submandibular and pancreatic acini loaded with the Ca²⁺-sensitive dye Fura-2 or the Na⁺-sensitive dye SBFI. Pancreatic acini showed no changes in Na during either transient or persistent changes in Ca. Increases in Ca produced by exposure of submandibular gland acini to carbachol, a muscarinic cholinergic agonist, were followed by an increase in Na after a delay of 5–10 s. When Ca²⁺ stores were mobilized without Ca²⁺ influx Na also increased, but in acini loaded with BAPTA, a nonfluorescent Ca²⁺ chelator, the transient increase in Ca²⁺ caused by mobilization of stored Ca²⁺ was virtually abolished, as was the increase in Na. In the presence of ionomycin, increases in Ca were followed by increases in Na. Ca²⁺-dependent increases in Na were abolished in Na⁺-free buffer and by the presence of furosemide, a blocker of Na⁺-K⁺-2Cl⁻ cotransport. In other studies, extracellular ATP (ATP_o) produced an increase in Ca and Na. The steady-state increase in Ca was reduced by increasing extracellular Na⁺ concentrations (Na) in dose-dependent fashion (IC₅₀ = 16.4 ± 4.7 mM Na⁺). Likewise, increasing Na reduced ATP_o-stimulated ⁴⁵Ca²⁺ uptake at steady state (IC₅₀ = 15.8 ± 9.2 mM Na⁺). Changing Na had no effect on carbachol-stimulated increases in Ca. We conclude that, in rat submandibular gland acini, ATP_o promotes an increase in Ca and Na via a common influx pathway and that, under physiologic conditions, Na⁺ significantly limits the ATP_o-stimulated increase in Ca. In the presence of carbachol, however, Na rises in Ca-dependent fashion in submandibular gland acini via stimulation of Na⁺-K⁺-2Cl⁻ cotransport. © 1996 Wiley-Liss, Inc.     

Apparent molar volumes of some amino acids and peptides in aqueous urea solutions

Densities of solutions of several α-amino acids and peptides in 3 and 6m aqueous urea solvents have been determined at 298.15 K. These data have been used to evaluate the infinite-dilution apparent molar volumes of the solutes and the volume changes due to transfer (V ) of the α-amino acids and peptides at infinite dilution from water to aqueous urea solutions. The sign and magnitude of the V values have been rationalized in the framework of Friedman's cosphere-overlap model. The V values for the glycyl group (? CH₂CONH? ) and alkyl side chains have been estimated.     

An Asymptotically Optimal Adaptive Selection Procedure in the Proportional Hazards Model with Conditionally Independent Censoring

Assume k independent populations are given which are distributed according to R, …,R (ϑ_i ∈ Θ ⊆ R ). Taking samples of size n the population with the smallest ϑ-value is to be selected. Using the framework of Le Cam's decision theory (Le Cam , 1986; Strasser , 1985) under mild regularity assumptions, an asymptotically optimal selection procedure is derived for the sequence of localized models. In the proportional hazards model with conditionally independent censoring, an asymptotically optimal adaptive selection procedure is constructed by substituting the unknown nuisance parameter by a kernel estimator.     

How Large is the Probability for the Estimate of a Variance Component to be Negative?

For a balanced one-way classification, where the normally distributed observations obey a random model y_ij=μ+b_i+c_ij with two variance components var (b_i) = δ and var (c_ij) = δ, the probability is given that the analysis of variance estimate of δ will be negative. This probability depends on δ/δ and the degrees of freedom in the ANOVA table. Tables for this probability are given. If the normally distributed observations obey an intra-class correlation model, the probability that the Mean Square between groups is smaller than the Mean Square within groups can also be evaluated from the given tables.     

On the blue color of triiodide ions in starch and starch fractions. I. Characterization and assignment of absorption and circular dichroism spectra of triiodide ions in amylose

Four fundamental Raman lines were observed at 159, 111, 55 and 27 cm^-1 corresponding to the I bound (I) in amyloses with DP from 20 to 100, regardless of the degree of polymerization of I and the excitation wavelength. The spectral resolution was based on the molar extinction coefficient and molar ellipticity spectra of I. Eight bands, named, S₁, S₂, ?, S₈ from long to short wavelength, were isolated. These were found regardless of the DP. By a resonance excitation Raman study, the characteristics of S₃ and S₄, comprising the shoulder around 480 nm, were found to be different from those of S₁ and S₂, comprising the blue band. The assignment of the spectra was based on the electronic states of the monomeric I in the exciton-coupled dimeric unit. It was concluded that the blue band (S₁,S₂) belonged to the long-axis transitions and the shoulder band (S₃,S₄) to the short-axis ones on the monmeric coordinate system.     

Conformation of poly(L-arginine). I. Effects of anions

The conformational transition of poly(L -agrignine) by binding with various mono-, di-, and polyvalent anions, especially with SO, was studied by CD measurements. The intramolecular random coil-to-α-helix conformational transition and the subsequent transition to the β-turn-like structure was caused by binding with SO. The binding data obtained from equilibrium dialysis experiments showed that the α-helical conformation of poly(L -arginine) is stabilized at a 1:3 stoichiometric ratio of bound SO to arginine residue; at higher free SO concentrations, the α-helix converts to the β-turn-like structure accompanied by a decrease in amount of bound SO. The same conformaitonal transition of poly(L -arginine) also occurred in the solutions of other divalent anions (SO, CO, and HPO) and polyvalent anions (P₂O, P₃O). Among the monovalent anions examined, CIO and dodecyl sulfate were effective in including α-helical conformation, while the other monovalent anions (OH^?, Cl^?, F^?, H₂PO, HCO and CIO) failed to induce poly(L -arginine) to assume the α-helical conformation. Thus, we noticed that, except for dodecyl sufate, the terahedral structure is common to the α-helix-forming anions. A well-defined model to the α-helical poly(L -arginine)/anion complex was proposed, in which both the binding stoichiometry of anions to the arginine residue and the tetrahedral structure of anions were taken into consideration. Based on these results, it was concluded that the tetrahedral-type anions stabilize the α-helical conformation of poly(L -arginine) by crosslinking between two guanidinium groups of nearby side chains on the same α-helix through the ringed structures stabilized by hydrogen bonds as well as by electrostatic interaction. Throughout the study it was noticed that the structural behavior of poly(L -arginine) toward anions is distinct from that of poly(L -lysine).     

Inorganic carbon sources and biomass regulation in intensive microalgal cultures

Three freshwater and one marine algal species were grown under inorganic carbon limitation in laboratory continuous cultures. Comparisons were made between HCO₃^? alkalinity and bubbled CO₂ as carbon sources. HCO₃^? alkalinity was an excellent source of inorganic carbon below specific pH levels, but chemical precipitation at high pH placed an upper limit on productivity that was far lower than potential light-limiting levels. With bubbled CO₂ it was possible to achieve light limitation. The main factor controlling productivity was the mass flux of inorganic carbon added to the culture, which is the product of gas flow rate and influent P level. Small bubbles were more efficient than large bubbles at low gas flow rates and P levels, but led to froth flotation of algal cells and concomitant reductions in productivity at high bubble rates. At 1% CO₂ productivity was still dependent on mass fluxes of added carbon, but was independent of bubble size. At high bubble rates with 1% CO₂ narcosis was evident. Maximum yields occurred at intermediate dilution rates when inorganic carbon was supplied via bubbled gas.     

Conformational analysis of acetyl-L-phenylalanine p-acetyl and p-valeryl anilides

Empirical force-field calculations and ir and ¹H-nmr spectra indicate that five-membered (C₅) and seven-membered (C) hydrogen-bonded rings are the preferred conformations of acetyl-L -Phe p-acetyl and p-valeryl anilides in nonpolar media. The C₅/C ratio was found to be dependent on the dryness of the solute and the solvent. This fact and the results from conformational-energy calculations suggest that a molecule of water participates in the stabilization of the C conformation.     

On the application of polyelectrolyte limiting laws to the helix-coil transition of DNA. IV. Depedence of helix stability on the concentration of divalent metal ions.

The molecular theory of the previous paper in this series is extended to determine the effect of divalent metal ions on helix stability relative to coil at fixed ionic strength and nucleotide phosphate concentration. Specification of the state of condensed counterions, as well as their concentration, is essential for the solution of this problem, and it is assumed that they translate freely within a thin cylindrical shell close to the polynucleotide. As a function of divalent counterion concentration m the relative stability of the helix is highly nonlinear. Although the overall trend is that the helix stability increases with addition of divalent metal ion, there is a narrow concentration range for which it decreases slightly. The behavior of the relative stability as a function of m is determined by the translational degrees of freedom of the counterions, both univalent and divalent, both condensed and uncondensed. Detailed comparison of the theory with data is not given here, but it is pointed out that the calculated values of the relative stability are consistent with the order of magnitude of the observed effect Mg²⁺ on the melting temperature.     

Hydrobiological Studies on Suraj Kund and Chandrama Kund,Hot Springs of Rajgir,Bihar, India

The hot water of Rajgir springs is used for drinking and bathing purposes by tourists. Certain physico-chemical characteristics (temperature, pH, NO, PO, etc.) of the water along with phycological parameters viz. community composition, species diversity, standing crop etc. were measured from June 1986 to April 1987. The water was deficient in Na, NO and PO ions. The hot springs were mainly dominated by algae belonging to Cyanophyceae and Bacillariophyceae. The algal community comprised 18 species, with dominance of Cyanophyceae over Bacillariophyceae. While Mastigocladus laminosus and species of Phormidium were dominant in Suraj Kund, species of Oscillaroria and Synechococcus dominated in Chandrama Kund. Diatoms comprised about 10 % of the algal community. Though there was a considerable seasonal change in species diversity of the algal community the total biomass (chlorophyll a extracted per unit area from the algal mat) remained constant.     

Conformation and folding in histones H1 and H5

Denatured histones H1 and H5 can be readily refolded on salt addition. Their digestion by trypsin leads to limit peptides of about 80 residues having the same nmr and CD spectra as those of the intact parent histones. Scanning microcalorimetry shows that (1) the folded structures of H1 and H5 are located entirely in their limit peptides; (2) both have values of the specific denaturation enthalpy typical for small globular proteins; and that (3) both exhibit a classic “2-state” transition (ΔH = ΔH). The heat-denaturation profiles of H5 measured using intrinsic and extrinsic Cotton effect and side-chain nmr peaks do not coincide at all. Only the intrinsic Cotton effects give a T_m and ΔH close to that from microcalorimetry. We conclude that these proteins exhibit large-scale side-chain motions that precede the macroscopic cooperative transition.     

Collapse of DNA caused by trivalent cations: pH and ionic specificity effects

A comparison of the condensation of T4 phage DNA by spermidine and Co(NH₃) at pH values between 5.1 and 10.2 has been made using quasielastic light scattering to determine translational diffusion coefficients and Stokes radii. Co(NH₃) is more effective than spermidine in causing condensation at all pH, indicating that the differences observed in previous work were not due to pH effects, as might have been inferred from recent theories of intermolecular forces. The DNA particles collapsed with Co(NH₃) are smaller than those obtained with spermidine. The hydrodynamic radius of spermide-collapsed structures decreases slightly with increasing pH, while the size of the Co(NH₃)collapsed structures is almost independent of pH. These results confirm that there are specific ion effects in DNA condensation by oligocations, in addition to the dominant general polyelectrolyte effects.     

Chemiluminescence of human bloodstream monocytes and neutrophils: An unusual oxidant(s) generated by monocytes during the respiratory burst

Maximal rates of O and H₂O₂ production by human bloodstream monocytes activated during the respiratory burst by phorbol ester were only about 10% of those of neutrophils. Furthermore, monocytes possess only about 5% of the myeloperoxidase activity of neutrophils and so can only produce low levels of HOCI and related compounds. These combined reductions in O generating ability and lower myeloperoxidase levels result in low luminol chemiluminescence stimulated during the respiratory burst of monocytes. However, although monocytes generate much lower levels of O and H₂O₂ than neutrophils, these cells produce comparable rates of PMA-stimulated lucigenin chemiluminescence. Hence, this assay does not accurately reflect the production of either of these two oxidants by activated phagocytes, and further lucigenin must react with some other oxidant(s) via a process which leads to photon emission. This oxidant(s) is not O, H₂O₂, · OH, ¹O₂ or NO, but is derived from O generated during the respiratory burst and is generated in greater quantities by activated monocytes compared with neutrophils. Thus, lucigenin chemiluminescence is an indirect measure of superoxide release.     

A stereospecific complex of poly(I) with ammonium ion

We describe conditions which lead to complete helix formation of poly(I) in the presence of NH. Binding of NH is shown to be specific in the presence of Li⁺, which does not by itself support helix formation under these conditions. The NH–poly(I) complex is characterized by uv, CD, and ir spectroscopy. The CD spectrum is strikingly different from those of the Na⁺ or K⁺ complexes, the first extremum being changed from negative for the metal ions to positive for NH. A stereospecific model is proposed for the NH–poly(I) helix in which the N of NH is located on the axis of the four-stranded helix, midway between planar tetramers formed by the bases. The model is consistent with the tetrahedral symmetry of NH, the requirement for four acceptable hydrogen bonds, the observed stability of the helix, and the accepted geometry of the backbone.     

Fed batch culture of Saccharomyces cerevisiae: A perspective of computer control to enhance the productivity in baker's yeast cultivation

A means to avoid the glucose effect in the production of baker's yeast from glucose and/or molasses in a fed batch culture by controlling the feed rate of fresh medium with an ad hoc measurement of the respiratory quotient, RQ, is presented. The feed rate is changed stepwise here such that the value of RQ ranges from 1.0 to 1.2 throughout the cultivation. Thus far, the specific growth rate based on the total cell mass and the growth yield obtained throughout are 0.24 hr^?1 and 0.55 g cell/g glucose. Prior to the experimental run mentioned above, equations to predetermine the feed rate and concentration of glucose in the feed are derived from the mass balance of limiting substrates (glucose). Since values of either RQ or I (Q x, oxygen consumption rate with respect to the total cell mass in the fermenter) can be measured quite easily and reliably, computer control of the fermentation in light of this information is discussed.     

Number of binding sites of DNA and polynucleotides with tris(2,2'-dipyridyl)ruthenium(II) cations

The binding of tris(2,2′-bipyridyl)ruthenium(II) cations [Ru(bpy)] with single- and double-stranded (ss and ds) DNA, and the polynucleotides poly(A), poly(C), poly(G), poly(I), poly(I) · poly(C), and poly(U), was studied in aqueous solution. Steady-state electrical conductivity measurements with the polynucleotides, ssDNA, and dsDNA reveal that approximately three nucleotides offer one binding site. This may be compared with the ratio [nucleotide]/[Mg²⁺] of 2.4 : 1 for dsDNA. After laser excitation (353 nm), the luminescence of Ru(bpy) bound to nucleic acids shows two decay components. The contribution of the fast component, which is interpreted as resulting from quenching processes of the absorbed ruthenium complex, exhibits a maximum with increasing [nucleotide]/[Ru(bpy)] at a ratio of about three to one. Bound Ru(bpy) can be released from the strand by addition of NaClO₄ [half-concentration: 2.5 and ≤ 10 mM for poly(U) and dsDNA, respectively].