Estimation of absorption efficiency in polychaetes using nitrogen content of food

From 14 values reported for nine species of polychaetes, a highly significant (P < 0.0005) multiple correlation (r = 0.987) between the nitrogen content of food (N) and absorption efficiency (Ae) is observed and is adequately fitted by the polynomial cubic equation: Ae = 11.744−13.353 N+6.510 N²−0.458 N³.     

Autocatalytic peroxidation of ferrocytochrome c

1. Ferricytochrome c acts as a catalyst in the peroxidation of ferrocytochrome c thereby giving rise to an autocatalytic reaction.

2. The rate of the peroxidation reaction is proportional to the concentration of H₂O₂ and ferricytochrome c but is independent of the concentration of ferrocytochrome c in the concentration ranges studied.

3. Integration of the rate equation, d[c³⁺]/dt = k[c³⁺][H₂O₂], gives a theoretical expression which fits the experimental time courses for the ferrocytochrome c peroxidation reaction.

4. No direct spectral evidence was found for the formation of a catalytically active ferricytochrome c-H₂O₂ derivative. Kinetic evidence is presented, however, which indicates the existence of such an intermediate.

5. Ferricytochrome c was more susceptible than ferrocytochrome c to an apparent degradation reaction caused by excess H₂O₂, thus supporting the idea that the cytochrome c heme iron is more accessible in the oxidized form.     

The effect of the trolox equivalent antioxidant capacity (TEAC) in plasma on the formation of 4-aminobiphenylhaemoglobin adducts in smokers

Smokers are exposed to a number of carcinogenic compounds including aromatic amines such as 4-aminobiphenyl. Antioxidants are thought to be involved in the defence against the damaging effect of such carcinogens. Recently it has been shown that plasma antioxidant status in smokers is diminished compared with non-smokers. In this study we investigated in 40 smokers whether the trolox equivalent antioxidant capacity (TEAC) in plasma could be quantitatively related to exposure to cigarette smoke. The biomarkers 4-aminobiphenylhaemoglobin (4-ABP-Hb) adduct and cotinine were determined as indices of cigarette smoke exposure. A correlation between 4-ABP-Hb adduct levels and plasma cotinine levels was found for the whole population studied, who smoked 4-70 cigarettes per day (n = 40, r² = 0.12, p = 0.03). A significant inverse relationship was found between TEAC and 4-ABP-Hb levels (n = 40, r² = 0.17, p = 0.008). Multiple regression analysis showed a strong relationship between 4-ABP-Hb levels and plasma TEAC and cotinine levels (n = 40, r² =0.29, p = 0.002). These findings provide strong evidence that the 4- ABP-Hb adduct represents a valuable biomarker of (internal) exposure to tobacco smoke, and also that the formation of this marker is dependent on the plasma antioxidant status. The multiple regression analysis results show that the measure of effect (4-ABP-Hb adduct formation) is largely determined by dose (cotinine) and protection (TEAC).     

Tree size and flowering intensity as affected by nitrogen fertilization in non-bearing orange trees grown under Mediterranean conditions

A field experiment was conducted in the South of Portugal with young (0-3 years old) non-bearing ‘Lane Late’ orange trees. Five nitrogen (N) rates were used in a randomised block design with three replicates. The 180 g N tree⁻¹ over three years led to the greatest canopy width (176 cm) and volume (2,697 dm³). The greatest rate applied (720 g N tree⁻¹ in the three years) led to the largest flower yield. Nitrogen concentration in the flowers significantly increased with fertilizer N, and also with the flowering period up to the 23^rd day, declining thereafter. Flower yield was strongly correlated (r = 0.99, p < 0.001) with flower N concentration. Nutrient composition of flowers and of mature leaves from the spring flush was compared. Significant correlations were found for N (r = 0.47, p <0.01), P (r = -0.49, p <0.01), K (r = 0.44, p <0.05) and Ca (r = 0.87, p <0.001), suggesting that flowers can be used as a tool to diagnose the nutritional status of trees. Canonical analysis (with N treatment as dummy-variables) showed strong relationships between canopy width and N, which were greater at the larger rates of fertilizer application, and strong and inverse relationships between K and Mg, also with the greatest N rates.     

3D-QSAR studies on natural acetylcholinesterase inhibitors of Sarcococca saligna by comparative molecular field analysis (CoMFA)

We have derived a comprehensive structure–activity relationship (SAR) picture for a new series of natural acetylcholinesterase inhibitors isolated from Sarcococca saligna. A set of 32 previously isolated and tested pregnane-type steroidal alkaloids inhibitors were investigated with respect to their IC₅₀ values (pIC₅₀) against the AChE enzyme in order to derive CoMFA models using atom-based alignment. A highly significant CoMFA model was obtained with r² value of 0.974. The q² (cross validation r²) value also confirms the statistical significance of our model.     

Relationship between cyprid energy reserves and metamorphosis in the barnacle Balanus amphitrite Darwin (Cirripedia; Thoracica)

Nauplii batch cultures of Balanus amphitrite were reared with four different diatoms (Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, silicate-limited C. gracilis) at three different cells concentrations: 1×10⁵, 5×10⁵, and 1×10⁶ cells ml⁻¹. The cyprid energy reserves were quantified as the ratio of triacylglycerols (TAG) to DNA. Energy reserves of larvae fed on different diatoms at a concentration of 1×10⁶ cells ml⁻¹ were ranked in the order: silicate-limited C. gracilis>C. gracilis>T. pseudonana>S. costatum. There was a significant linear relationship between the TAG content of the diet and cyprid energy reserves. The effect of cyprid energy reserves on metamorphosis to polystyrene surface in the presence and the absence of conspecific settlement factor (SF) was studied after 12, 24, and 48 h of incubation. A strong positive correlation between energy reserves and percent metamorphosis was observed in the absence of SF (r_{12 h}=0.88, r_{24 h}=0.82, r_{48 h}=0.68, P<0.05). A weak positive correlation was observed in the presence of SF (r_{12 h}=0.43, r_{24 h}=0.48, r_{48 h}=0.50, P<0.05). In both treatments, more than 80% of the cyprids with high energy reserves metamorphosed within 24 h. In contrast, a high proportion of cyprids with low energy reserves metamorphosed in response to SF in 24 h. Our results indicate that discriminatory metamorphic behavior of cyprids is closely linked to their TAG/DNA ratio, a proxy for energy reserve.     

A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus

1. Single reduced methyl viologen (MV^.+) acts as an electron donor in a number of enzyme systems. The large changes in extinction coefficient upon oxidation (λ_max 600 nm; MV^.+, = 1.3 · 10⁴ M⁻¹ · cm⁻¹; oxidised form of methyl viologen (MV²⁺), = 0.0) make it ideally suited to kinetic studies of electron transfer reactions using stopped-flow and standard spectrophotometric techniques.

2. A convenient electrochemical preparation of large amounts of MV^.+ has been developed.

3. A commercial stopped-flow apparatus was modified in order to obtain a high degree of anaerobicity.

4. The reaction of MV^.+ with O₂ produced H₂O₂ (k > 5 · 10⁶ M⁻¹ · s⁻¹, pH 7.5, 25 °C). H₂O₂ subsequently reacted with excess MV^.+ (k = 2.3 · 10³ M⁻¹ · s⁻¹, pH 7.5, 25 °C) to produce water. The kinetics of this reaction were complex and have only been interpreted over a limited range of concentrations.

5. The results support the theory that the herbicidal action of methyl viologen (Paraquat, Gramoxone) is due to H₂O₂ (or radicals derived from H₂O₂) induced damage of plant cell membrane.     

The activation by Ca of the ATPase of extracted muscle fibrils with variation of ionic strength, pH and concentration of MgATP

1. The alteration of the Ca²⁺ requirements of the ATPase activity of fibrils from rabbits and crabs at varying ionic strength, pH and concentration of MgATP (i.e. MgATP²⁻ + MgHATP⁻) was investigated.

2. Under physiological conditions, it was found that the ATPase activity of rabbit and crab fibrils after an initial increase decreased steeply when the Ca²⁺ concentration is raised above 1×10⁻⁴ M. This is a primary effect of the over-optimal Ca²⁺ concentration and not a secondary one caused by the influence of accompanying ions.

3. The Ca²⁺ requirements for ATP splitting by rabbit fibrils remain constant at an ionic strength from 0.1 to 0.2 and for a MgATP concentration in the range from 0.5 to 10 mM. At I = 0.05 it is about 5 times smaller than at 0.1. When the pH is decreased from 8 to 7, the Ca²⁺ requirements are increased some 10 times but only 3 times when the pH is varied between 7 and 6.

4. In crab fibrils, there is no alteration of the Ca²⁺ requirements when the ionic strength is varied between 0.05 and 0.2, but a reduction of the pH from 8.0 to 6.0 raises the Ca²⁺ requirements for half activation and for threshold by a factor of 10. Changing the MgATP concentration increases the Ca²⁺ requirements only in the range from 1 to 5 mM, while the concentration required in 0.5 mM is identical with that at 1 mM, and 10 mM corresponds to 5 mM.

5. It can be deduced from the experimental results that at a pH above 6.0 maximal activation is always obtained if the Ca²⁺ concentration is 5×10⁻⁵ M. By contrast, relaxation is only achieved when the Ca²⁺ concentration is below 1×10⁻⁷ M for pH 7.0 and I > 0.1 or below 1×10⁻⁸ for pH > 7.0 or I < 0.1.

6. To achieve complete relaxation, an ethyleneglycoldiaminotetraacetate (EGTA) concentration of 1 mM is sufficient, even when there is a large degree of contamination by Ca²⁺ as long as the pH stays above 6.5.     

Tricarbonyl(1,10-phenanthroline) (imidazole) rhenium(I): a powerful photooxidant for investigations of electron tunneling in proteins

The structure of [Re(CO)₃(phen)(im)]₂SO₄·4H₂O has been determined by X-ray crystallography. The yellow crystals are orthorhombic, space group Pccn (No. 56), with a=17.456(6), B=18.194(5), C=12.646(4) Å, R=0.063 for F_o²>0, R=0.032 for F_o²>3σ. The compound, which also has been characterized by IR, ¹H NMR, and UV---Vis spectroscopies, exhibits room temperature luminescence in aqueous solution (τ=120 ns) as well as reversible oxidation and reduction in acetonitrile solution (1.85 and −1.30 V versus SCE). The redox properties of the excited state of the complex (E⁰(Re^+*/0 = 1.2; E⁰(Re^2+/+*) = −0.7 V) are being exploited in studies of laser-induced electron tunneling in Re(CO)₃(phen)(histidine)-modified proteins.     

Effects of season on the yield and quality of agar from Gracilaria species (Gracilariaceae, Rhodophyta)

The effect of season on yield and physical properties of agars extracted from Gracia gracilis and G. bursa-pastoris were determined. The agar yield from G. gracilis was maximum during spring (30%) and minimum during autumn (19%). In G. bursa-pastoris, the agar yield was greatest in summer (36%) and lowest in winter (23%). Agar yield from G. bursa-pastoris was positively correlated with temperature (r=0.94; P<0.01) and salinity (r=0.97; P<0.01) and negatively with nitrogen content (r=−0.93; P<0.01). Agar gel strengths fluctuated from 229 to 828 g cm⁻² and 23 to 168 g cm⁻² for G. gracilis and G. bursa-pastoris, respectively. The gelling temperature showed significant seasonal variation for both species. Chemical analysis of agar from the two seaweeds indicated variation in 3,6-anhydrogalactose and sulfate content (P<0.01). Furthermore, there was an inverse correlation between the two chemical variables. In general, agar extracted from G. gracilis possessed better qualities than agar extracted from G. bursa-pastoris and can be considered a candidate for industrial use.     

Biochemical and biophysical studies on cytochrome c oxidase XV. Reaction with fluoride

1. Fluoride is a mixed-type inhibitor of the cytochrome c oxidase activity with a K_i for the free enzyme of 10 mM and a K_i for the cytochrome c-complexed enzyme of 35 mM.

2. Fluoride shifts the γ-band of the enzyme from 423 to 421 nm and the -band from 597 to 598 nm. The difference spectrum (oxidized enzyme in the presence of fluoride minus oxidized enzyme) has peaks at 400, 453, 482, 605 and 638 nm and troughs at 430, 520, 552 and 674 nm. The changes in absorbance are small (about 3% at absorbance maxima) with respect to those of other hemoproteins.

3. On addition of fluoride to isolated cytochrome c oxidase 3 reactions can be distinguished: (I) a bimolecular binding reaction (K_on = 4 M⁻¹ · s⁻¹ and k_off = 2.9 · 10⁻²s⁻¹ at 25 °C, pH 7.4) contributing at 638 nm and 430 nm; (II) a first-order reaction (k = 2.4 · 10⁻²) s⁻¹ at 22 °C, pH 7.2) visible mainly at 430 nm and (III) a very slow reaction with a half-time in the order of 10 min.

4. The spectroscopic dissociation constants for the fluoride binding, determined from Hill plots using the absorbance changes at 638 and 430 nm, are similar (7 and 10 mM, respectively, at 22 °C, pH 7.2).

5. A mechanism for the reaction is discussed in which the bimolecular binding reaction is followed by a conformational change of the enzyme-fluoride complex.     

Viscous energy dissipation in a model of the human bronchial tree

The viscous energy dissipation in a two generation model of the human bronchial tree is determined from inspiratory velocity and static pressure data obtained for large Reynolds numbers (10⁴ < Re < 10⁵). This dissipation is found to be an increasing function of both Re and distance downstream from the inlet of the model. The ratio of the dissipation in the model to the energy dissipation in an equivalent straight pipe system is determined. This ratio, Z^*, for the model is compared to values in the literature for lower (laminar) Re. There is more dissipation in the branched model than in a straight pipe (Z^* > 1) and turbulence keeps Z^* at roughly a fixed value for large Reynolds numbers (10⁴ < Re < 10⁵). Z^* values for curved pipes are also compared to the branching system values. It is found that the energy dissipation for the branched model behaves similarly to that in curved pipes.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Biochemical and biophysical studies on cytochrome c oxidase XIV. The reaction with cytochrome c as studied by pulse radiolysis

1. The reduction of cytochrome c oxidase by hydrated electrons was studied in the absence and presence of cytochrome c.

2. Hydrated electrons do not readily reduce the heme of cytochrome c oxidase. This observation supports our previous conclusion that heme a is not directly exposed to the solvent.

3. In a mixture of cytochrome c and cytochrome c oxidase, cytochrome c is first reduced by hydrated electrons (k = 4 · 10¹⁰ M⁻¹ · s⁻¹ at 22 °C and pH 7.2) after which it transfers electrons to cytochrome c oxidase with a rate constant of 6 · 10⁷ M⁻¹ · s⁻¹ at 22 °C and pH 7.2.

4. It was found that two equivalents of cytochrome c are oxidized initially per equivalent of heme a reduced, showing that one electron is accepted by a second electron acceptor, probably one of the copper atoms of cytochrome c oxidase.

5. After the initial reduction, redistribution of electrons takes place until an equilibrium is reached similar to that found in redox experiments of Tiesjema, R. H., Muijsers, A. O. and Van Gelder, B. F. (1973) Biochim. Biophys. Acta 305, 19–28.     

Total gene deletions and mutant frequency of the HPRT gene as indicators of radiation exposure in Chernobyl liquidators

This study was conducted to determine the utility of deletion spectrum and mutant frequency (MF) of the hypoxanthine phosphoribosyl transferase gene (HPRT) as indicators of radiation exposure in Russian Liquidators who served in 1986 or 1987 in the clean up effort following the nuclear power plant accident at Chernobyl. HPRT MF was determined using the cloning assay for 117 Russian Controls and 122 Liquidators whose blood samples were obtained between 1991 and 1998. Only subjects from whom mutants were obtained for deletion analysis are included. Multiplex PCR analysis was performed on cell extracts of 1080 thioguanine resistant clones from Controls and 944 clones from Liquidators. Although the deletion spectra of Liquidators and Controls were similar overall, the Liquidator deletion spectrum was heterogeneous over time. Most notable, the proportion of total gene deletions was higher in 1991–1992 Liquidators than in Russian Controls (χ²=10.5, p=0.001) and in 1993–1994 Liquidators (χ²=8.3, p=0.004), and was marginally elevated relative to 1995–1996 Liquidators (χ²=3.3, p=0.07). This type of mutation has been highly associated with radiation exposure. Total gene deletions were not increased after 1992. Band shift mutations were also increased in the 1991–1992 Liquidators but were associated with increased MF of both Liquidators and Controls (p=0.009), not with increased MF in 1991–1992 Liquidators (p=0.7), and hence are not believed to be associated with radiation exposure. Regression analysis demonstrated that relative to Russian Controls HPRT MF was elevated in Liquidators overall when adjusted for age and smoking status (37%, p=0.0001), and also was elevated in Liquidators sampled in 1991–1992 (72%, p=0.0076), 1993–1994 (22%, p=0.037), and 1995–1996 (62%, p=0.0001). In summary, HPRT MF was found to be the more sensitive and persistent indicator of radiation exposure, but the specificity of total gene deletions led to detection of probable heterogeneity of radiation exposure within the exposed population.     

The respiratory chain of Azotobacter vinelandii II. The effect of cyanide on cytochrome d

1. Cyanide causes a slow disappearance of the oxidized band (648 nm) of cytochrome d in particles of Azotobacter vinelandii and inhibits the appearance of the reduced band (631 nm). No effect of cyanide is found on the reduced band of cytochrome d.

2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide deviates from first-order kinetics at lower temperatures (22 °C) indicating that at least two conformations of the enzyme are involved. At higher temperatures (32 °C) the observed kinetics of the cyanide reaction are first order with a k_on = 0.7 M⁻¹·s⁻¹ and with an estimated k_off of approximately 5·10⁻⁵ s⁻¹.

3. The value of the k_off (7·10⁻⁴−14·10⁻⁴ s⁻¹ at 32 °C) determined from the rate of reduction of cyanocytochrome d by Na₂S₂O₄ or NADH is one order of magnitude larger than the k_off value found when the enzyme is in its oxidized state.

4. No effect of cyanide is found on the spectrum of cytochrome a₁.     

Metal ion substitution in phospholipase C (Bacillus cereus) and its effect on enzyme stability

The effect of guanidinium chloride solutions on the circular dichroism of native (ZnZn-) and apophospholipase C (Bacillus cereus) indicated marked protein unfolding at denaturant concentrations of 1.4–1.8 M and 0.1–0.6 M, respectively. With the apoenzyme near u.V. region circular dichroism bands remained even after all ordered structure appeared to have been lost. Apophospholipase C bound two equivalents of Ni²⁺, Cd²⁺, Co²⁺, Mn², Pb²⁺ or Cu²⁻, with only the latter metal causing marked changes either in circular dichroism or protein fluorescence relative to the native enzyme. Stability in guanidinium chloride for the metalloforms of phospholipase C decreased in the order: ZnZn->ZnCo->NiNi->CoCo->PbPb->CdCd->MnMn-apoenzyme.     

Hydrodynamic properties of the fractions of mannan formed by Rhodotorula rubra yeast

Aqueous solutions of fractions of an extracellular linear mannan formed by Rhodotorula rubra yeast have been investigated by hydrodynamic methods (high-speed sedimentation, translation isothermic diffusion and viscometry). The molecular weight was determined according to Svedberg ( ) and the polydispersity parameters of the initial sample were also determined (M_w/M_n = 1·20 and M_z/M_w = 1·21). Relationships between the molecular weight (M) and s_o, D_o and [η] in the range were: [η] = 2·33 × 10⁻² M^0.75, D_o = 1·65 × 10⁻⁴ M^0·58, s_o = 2·24 × 10⁻¹⁵ M^0·43. The equilibrium rigidity and hydrodynamic diameter of chains representing mannan molecules were evaluated.