Highly Sensitive Detection of Melamine Using a One-Step Sample Treatment Combined with a Portable Ag Nanostructure Array SERS Sensor

There is an urgent need for rapid and reliable methods able to detect melamine in animal feed. In this study, a quick, simple, and sensitive method for the determination of melamine content in animal feed was developed using surface-enhanced Raman spectroscopy on fabricated Ag nanorod (AgNR) array substrates with a one-step sample extraction procedure. The AgNR array substrates washed by HNO₃ solvent (10⁻⁷ M) and methanol and showed the good stability within 6 months. The Raman shift at △ν = 682 cm⁻¹ was used as the characteristic melamine peak in the calculations. Sufficient linearity was obtained in the 2–200 μg·g⁻¹ range (R² = 0.926). The limits of detection and quantification were 0.9 and 2 μg·g⁻¹, respectively. The recovery rates were 89.7–93.3%, with coefficients of variation below 2.02%. The method showed good accuracy compared with the tradition GC-MS analysis. This new protocol only need 2 min to fininsh the detection which could be developed for rapid onsite screening of melamine contamination in quality control and market surveillance applications.     

Omapatrilat,an Angiotensin-Converting Enzyme and Neutral Endopeptidase Inhibitor,Attenuates Early Atherosclerosis in Diabetic and in Nondiabetic Low-Density Lipoprotein Receptor–Deficient Mice

Omapatrilat inhibits both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP). ACE inhibitors have been shown to inhibit atherosclerosis in apoE-deficient mice and in several other animal models but failed in low-density lipoprotein (LDL) receptor– deficient mice despite effective inhibition of the reninangiotensin- aldosterone system. The aim of the present study was to examine the effect of omapatrilat on atherogenesis in diabetic and nondiabetic LDL receptor–deficient mice. LDL receptor–deficient male mice were randomly divided into 4 groups (n = 11 each). Diabetes was induced in 2 groups by low-dose STZ, the other 2 groups served as nondiabetic controls. Omapatrilat (70 mg/kg/day) was administered to one of the diabetic and to one of the nondiabetic groups. The diabetic and the nondiabetic mice were sacrificed after 3 and 5 weeks, respectively. The aortae were examined and the atherosclerotic plaque area was measured. The atherosclerotic plaque area was significantly smaller in the omapatrilat-treated mice, both diabetic and nondiabetic, as compared to nontreated controls. The mean plaque area of omapatrilattreated nondiabetic mice was 9357 ± 7293 μm², versus 71977 ± 34610 μm² in the nontreated mice (P = .002). In the diabetic animals, the plaque area was 8887 ± 5386 μm² and 23220 ± 10400 μm², respectively for treated and nontreated mice (P = .001). Plasma lipids were increased by omapatrilat: Meanplasma cholesterol in treated mice, diabetic and nondiabetic combined, was 39.31 ± 6.00 mmol/L, versus 33.12 ± 7.64 mmol/L in the nontreated animals (P = .008). The corresponding combined mean values of triglycerides were 4.83 ± 1.93 versus 3.00 ± 1.26 mmol/L (P = .02). Omapatrilat treatment did not affect weight or plasma glucose levels. Treatment with omapatrilat inhibits atherogenesis in diabetic as well as nondiabetic LDL receptor–deficient mice despite an increase in plasma lipids, suggesting a direct effect on the arterial wall.     

A study of some factors that influence the iodination of ox insulin

1. The influence of carrier iodide, iodine monochloride and pH on the labelling of ox insulin with ¹³¹I by the iodine monochloride method have been studied. 2. The quantitative effect of the iodide in the radioactive iodine preparation was that predicted from a calculation of its specific activity. No other interfering factors were detected in the [¹³¹I]iodide solutions used. 3. Increasing the molar ratio of iodine monochloride to insulin resulted in an increase followed progressively by a decrease in the proportion of ¹³¹I bound, while the total iodine bound increased to an amount characteristic of pH and thereafter remained constant. 4. The influence of pH on the iodination of insulin with iodine monochloride was complex and the pH curve showed two maxima, at pH2·8 and 6·4. At pH2·8 it was not possible to exceed 8 atoms of iodine bound per molecule by increasing the molar ratio of iodine monochloride. Similarly, at pH6·4 the substitution value of 11·5 atoms of iodine per molecule could not be exceeded. 5. Iodinated insulins containing an average of 1·96, 2·74, 6·0 and 7·0 atoms of iodine per molecule fully retained the ability to bind guinea-pig anti-(ox insulin) serum, and the ability to compete with unlabelled insulin for antibody sites only became significantly changed in the most highly substituted preparations and in the presence of large concentrations of unlabelled insulin. 6. The method for the iodination of insulin with 98% incorporation of ¹³¹I by using chloramine-t is described. 7. ¹³¹I-iodinated insulin prepared with graded quantities of chloramine-t in excess of that required for efficient labelling was less efficiently bound by guinea-pig anti-(ox insulin) serum than insulin labelled by the iodine monochloride method.     

myo-Inositol homeostasis in foetal rabbit lung

In several species, lung maturation is accompanied by a decline in the phosphatidylinositol content of lung surfactant and a concomitant increase in its phosphatidylglycerol content. To examine the possibility that this developmental change is influenced by the availability of myo-inositol, potential sources of myo-inositol for the developing rabbit lung were investigated. On day 28 of gestation the myo-inositol content of foetal rabbit lung tissue (2.3±0.5μmol/g of tissue) was not significantly different from that of adult lung tissue but the activity of d-glucose 6-phosphate:1l-myo-inositol 1-phosphate cyclase (cyclase) in foetal lung tissue (81.0±9.0nmol·h⁻¹·g of tissue⁻¹) was higher than that found in adult lung tissue (23.2±1.0nmol·h⁻¹·g of tissue⁻¹). Day 28 foetal rabbit lung tissue was found also to take up myo-inositol by a specific, energy-dependent, Na⁺-requiring mechanism. Half-maximal uptake of myo-inositol by foetal rabbit lung slices was observed when the concentration of myo-inositol in the incubation medium was 85μm. When the myo-inositol concentration was 1mm (but not 100μm) the addition of glucose (5.5mm) stimulated myo-inositol uptake. myo-Inositol uptake was observed also in adult rabbit lung and was found to be sub-maximal at the concentration of myo-inositol found in adult rabbit serum. The concentration of myo-inositol in the serum of pregnant adult rabbits (47.5±5.5μm) was significantly lower than that of non-pregnant adult female rabbits (77.9±9.2μm). On day 28 of gestation the concentration of myo-inositol in foetal serum (175.1±12.0μm) was much less than on day 25, but more than that found on day 30. A transient post-partum increase in the concentration of myo-inositol in serum was followed by a rapid decline. Much of the myo-inositol in foetal rabbit serum probably originates from the placenta, where on day 28 of gestation a high cyclase activity (527±64nmol·h⁻¹·g of tissue⁻¹) was measured. The gestational decline in serum myo-inositol concentration, together with the decreasing cyclase activity of the lungs, is consistent with the view that maturation of the lungs is accompanied by decreased availability of myo-inositol to this tissue.     

A new species of plasmodiidae (Coccidia: Hemosporidia) from the blood of the skink Scincus hemprichii (Scincidae: Reptilia) in Saudi Arabia

Fallisia arabica n. sp. was described from peripheral blood smears of the Skink lizard, Scincus hemprichii from Jazan Province in the southwest of Saudi Arabia. Schizogony and gametogony take place within neutrophils in the peripheral blood of the host. Mature schizont is rosette shaped 17.5 ± 4.1 × 17.0 ± 3.9 μm, with a L/W ratio of 1.03(1.02–1.05) μm and produces 24(18–26) merozoites. Young gametocytes are ellipsoidal, 5.5 ± 0.8 × 3.6 ± 0.5 μm, with a L/W of 1.53(1.44–1.61) μm. Mature macrogametocytes are ellipsoidal, 9.7 ± 1.2 × 7.8 ± 1.0 μm, with a L/W of 1.24(1.21–1.34) μm and microgametocytes are ellipsoidal, 7.0 ± 1.1 × 6.8 ± 0.9 μm. with a L/W of 1.03(1.01–1.10) μm. In comparison to the described Fallisia species, this new taxon has rosette schizonts and is larger than F. dominicensis, in Hispaniola, F. bipocrati, F. poecilopi, in Panama, F. thecadactyli in Venezuela, and F. effusa, F. simplex, F. modesta, in Brazil. F. arabica has fewer merozoites than F. effusa, F. poecilopi, F. thecadactyli and F. siamense in Thailand. This new species has more merozoites than F. dominicensis and F. modesta. All of these species belong to diverse saurian families (Agamidae, Gekkonidae, Polychrotidae, Scincidae and Teiidae) parasitize only thrombocytes or lymphocytes and some species parasitize immature erythroid cells and leucocytes.     

Rates of entry and oxidation of acetate,glucose, d(?)-β-hydroxybutyrate,palmitate, oleate and stearate,and rates of production and oxidation of propionate and butyrate in fed and starved sheep

1. Rates of entry and oxidation of a range of metabolites have been measured in tracheostomized sheep (diet, 800g. of lucerne chaff and 100g. of maize/day) by combining isotope-dilution techniques with the continuous measurement of total respiratory gas exchange, and ¹⁴CO₂ production during the intravenous or intraruminal infusion of ¹⁴C-labelled substrates. 2. Mean entry rates in fed and starved (24hr.) sheep respectively, expressed as mg./min./kg. body wt.^0·75, were: glucose, 5·0 (range 4·8–5·1, 2 observations) and 3·8 (3·2–4·2, 4); acetate, 10·8 (9·1–13·5, 4) and 5·8 (1); d(−)-β-hydroxybutyrate, 1·4 (1) and 1·5 (0·8–2·4, 4); palmitate, oleate and stearate (starved sheep only) 1·0 (0·6–1·9, 7), 0·9 (0·2–1·6, 10) and 0·9 (0·5–1·1, 11) respectively. 3. Production rates of propionate and butyrate in continuously feeding sheep were 6·4 (4·7–8·3, 4) and 4·3 (3·4–6·1, 4) mg./min./kg.^0·75 respectively, and in starved (24hr.) sheep were 2·5 (2·2–2·9, 2) and 1·0 (0·8–1·2, 2) mg./min./kg.^0·75 respectively. 4. Calculated terminal values for the specific radioactivity of respiratory ¹⁴CO₂ during measurements of entry rates and production rates were used to calculate the contributions of individual substrates to overall oxidative metabolism. Mean values for fed and starved sheep respectively were: glucose, 9·1 (8·6–9·6, 2) and 11·2 (5·9–15·1, 4)%; acetate, 31·6 (26·8–38·1, 4) and 22·1 (1)%; d(−)-β-hydroxybutyrate, 10·4 (1) and 4·8 (1·9–7·7, 4)%; propionate, 23·0 (13·8–29·9, 4) and 7·1 (6·8–7·4, 2)%; butyrate, 16·5 (13·7–20·5, 4) and 5·3 (5·2–5·3, 2)%; palmitate, oleate and stearate (starved sheep only), 4·7 (2·0–7·7, 7), 4·0 (1·2–6·6, 10) and 4·4 (3·8–5·8, 9)% respectively. The sum of these values for individual substrates in fed and starved sheep, excluding that of β-hydroxybutyrate and after correction of the glucose value for the known interrelations of this substrate with propionate, accounted for 76% and 58% respectively of total production of carbon dioxide. 5. Calculations based on the proportion of substrate entry directly oxidized indicated that the substrates studied accounted for 63% (fed sheep) and 43% (starved sheep) of total energy expenditure measured by oxygen uptake. The contribution of β-hydroxybutyrate was excluded, and corrections were made for glucose–propionate interrelations, and for the different rates of oxidation of the methyl and carboxyl fragments of acetate. 6. The present results have been combined with those obtained earlier in this Laboratory to examine the relationships between rates of substrate entry and oxidation, and concentrations of substrate in blood. Rates of entry of acetate, glucose, d(−)-β-hydroxybutyrate, palmitate and oleate (but not stearate) were well correlated with concentration in blood, and substrate contribution to production of carbon dioxide showed a similar correlation to blood concentration, except with glucose. 7. It was concluded that the general technique is of potential value in providing valid quantitative parameters of animal metabolism.     

Laser activation of rapid absorption changes in spinach chloroplasts and chlorella

The kinetics of the 520 mμ absorption change in spinach chloroplasts and Chlorella vulgaris following a flash from the ruby laser have been determined as follows: rise halftime ≤ 0.3 × 10⁻⁶ second; rapid recovery halftime = 5 to 6 × 10⁻⁶ second; intermediate recovery halftime = 4 × 10⁻⁴ second (spinach chloroplasts only); slow recovery halftime = 12 to 170 × 10⁻³ second, dependent on the measuring light intensity and aerobicity of the suspension.

The rapid phase of the 520 mμ reaction is approximately independent of temperature, from 295° to 77° Absolute.

With increasing oxygenation of the sample, the extent of the rapid phase decreases, the extent of the slow phase increases, while the extent of the intermediate phase in spinach chloroplasts remains constant.

In spinach chloroplasts, no recovery halftime of the 3 recovery phases for the 520 mμ absorption change was observed to correspond to the halftime for oxidation of cytochrome f (t_½ = 1.3 × 10⁻³ second).

     

Kinetics of thiamin cleavage by sulphite

Results are presented on the rate of thiamin cleavage by sulphite in aqueous solutions as affected by temperature (20–70°), pH(2·5–7·0), and variation of the concentration of either thiamin (1–20μm) or sulphite (10–5000μm as sulphur dioxide). Plots of the logarithm of percentage of residual thiamin against time were found to be linear and cleavage thus was first-order with respect to thiamin. At pH5 the rate was also found to be proportional to the sulphite concentration. In the pH region 2·5–7·0 at 25° the rate constant was 50m⁻¹hr.⁻¹ at pH5·5–6·0, and decreased at higher or lower pH values. The rate of reaction increased between 20° and 70°, indicating a heat of activation of 13·6kcal./mole.     

Plasma TSH and Serum T-4 Levels in Long-term Follow-up of Patients Treated with 131I for Thyrotoxicosis

In February 1972 58% of patients euthyroid after iodine-131 therapy given for thyrotoxicosis between 1954 and 1966 had a high plasma TSH (>7·4 μU/ml) and 42% a normal plasma TSH level. A group of 69 of the euthyroid patients with high plasma TSH levels (25·0±2·0 μU/ml) in 1972 were re-examined 15 and 24 months later. The mean plasma TSH in the 66 patients remaining euthyroid at 15 months was 22·6±1·8 μU/ml, while three patients had become hypothyroid. At 24 months 64 of the patients were still available for study, of whom 61 remained euthyroid with a mean plasma TSH of 21·6±2·0 μU/ml, and a further three had become hypothyroid.All of a group of 61 of the euthyroid patients with normal plasma TSH levels (4·0±0·2 μU/ml) in 1972 remained euthyroid at 24 months with a mean plasma TSH of 4·1±0·3 μU/ml, though the plasma TSH level had become slightly raised in three.The mean serum T-4 level in the euthyroid patients with a high plasma TSH was significantly lower, though still in the normal range, than that in the euthyroid patients with a normal plasma TSH both in 1972 and in 1974.Since no patient with a normal plasma TSH level after iodine-131 treatment six to 18 years earlier for thyrotoxicosis developed hypothyroidism over a two-year period, the follow-up of such patients need not be so rigorous as that of similarly treated euthyroid patients with raised plasma TSH levels in whom hypothyroidism developed at the rate of 5% per year.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

Glucose metabolism in the superovulated rat ovary in vitro. Effects of luteinizing hormone and the role of glucose metabolism in steroidogenesis

1. Superovulated rat ovary slices from rats treated with 20μg. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Δ⁴-3-oxo steroids (0·2μmole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90·0±4·6μmoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78·0±2·9μg. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U-¹⁴C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60·7±0·9, 2·4±0·1, 18·0±1·1 and 0·7±0·1μg. atom of carbon/g. wet wt./hr. and accounted for 104·5±1·9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [¹⁴C]carbon dioxide were increased by approx. 25%, and 108·4±3·2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the ¹⁴C incorporated into this fraction during incubation with [U-¹⁴C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of ¹⁴C were incorporated into these lipid fractions from [1-¹⁴C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [¹⁴C]lactate and glucose 6-phosphate (C-1) derived from [1-¹⁴C]-, [6-¹⁴C]- and [U-¹⁴C]-glucose, and the ratio of [¹⁴C]carbon dioxide yields from [1-¹⁴C]glucose and [6-¹⁴C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of ¹⁴C from [1-¹⁴C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [³H]water, [¹⁴C]sorbitol and glucose (1mg./ml.), the total water space (865±7·1μl./g.) and the extracellular water space (581±22μl./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540±23·6μl./g. to 639±31·3μl./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake.     

A Novel GH7 Endo-β-1,4-Glucanase from Neosartorya fischeri P1 with Good Thermostability,Broad Substrate Specificity and Potential Application in the Brewing Industry

An endo-β-1,4-glucanase gene, cel7A, was cloned from the thermophilic cellulase-producing fungus Neosartorya fischeri P1 and expressed in Pichia pastoris. The 1,410-bp full-length gene encodes a polypeptide of 469 amino acids consisting of a putative signal peptide at residues 1–20, a catalytic domain of glycoside hydrolase family 7 (GH7), a short Thr/Ser-rich linker and a family 1 carbohydrate-binding module (CBM 1). The purified recombinant Cel7A had pH and temperature optima of pH 5.0 and 60°C, respectively, and showed broad pH adaptability (pH 3.0–6.0) and excellent stability at pH3.0–8.0 and 60°C. Belonging to the group of nonspecific endoglucanases, Cel7A exhibited the highest activity on barley β-glucan (2020 ± 9 U mg^–1), moderate on lichenan and CMC-Na, and weak on laminarin, locust bean galactomannan, Avicel, and filter paper. Under simulated mashing conditions, addition of Cel7A (99 μg) reduced the mash viscosity by 9.1% and filtration time by 24.6%. These favorable enzymatic properties make Cel7A as a good candidate for applications in the brewing industry.     

L-Type Ca2+ Channel Sparklets Revealed by TIRF Microscopy in Mouse Urinary Bladder Smooth Muscle

Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca²⁺, referred to as Ca²⁺ sparklets have been detected in an intact detrusor smooth muscle electrical syncytium using a TIRF microscopy Ca²⁺ imaging approach. Sparklets were virtually abolished by the removal of extracellular Ca²⁺ (0.035±0.01 vs. 0.23±0.07 Hz/mm²; P<0.05). Co-loading of smooth muscle strips with the slow Ca²⁺ chelator EGTA-AM (10 mM) confirmed that Ca²⁺ sparklets are restricted to the cell membrane. Ca²⁺ sparklets were inhibited by the calcium channel inhibitors R-(+)-Bay K 8644 (1 μM) (0.034±0.02 vs. 0.21±0.08 Hz/mm²; P<0.05), and diltiazem (10 μM) (0.097±0.04 vs. 0.16±0.06 Hz/mm²; P<0.05). Ca²⁺ sparklets were unaffected by inhibition of P2X₁ receptors α,β-meATP (10 μM) whilst sparklet frequencies were significantly reduced by atropine (1 μM). Ca²⁺ sparklet frequency was significantly reduced by PKC inhibition with Gö6976 (100 nM) (0.030±0.01 vs. 0.30±0.1 Hz/mm²; P<0.05), demonstrating that Ca²⁺ sparklets are PKC dependant. In the presence of CPA (10 μM), there was no apparent change in the overall frequency of Ca²⁺ sparklets, although the sparklet frequencies of each UBSM became statistically independent of each other (Spearman''s rank correlation 0.2, P>0.05), implying that Ca²⁺ store mediated signals regulate Ca²⁺ sparklets. Under control conditions, inhibition of store operated Ca²⁺ entry using ML-9 (100 μM) had no significant effect. Amplitudes of Ca²⁺ sparklets were unaffected by any agonists or antagonists, suggesting that these signals are quantal events arising from activation of a single channel, or complex of channels. The effects of CPA and ML-9 suggest that Ca²⁺ sparklets regulate events in the cell membrane, and contribute to cytosolic and sarcoplasmic Ca²⁺ concentrations.     

The inhibition of β-lactamases from Gram-negative bacteria by clavulanic acid

The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t_½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t_½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m⁻¹·s⁻¹) to produce a relatively stable complex (t_½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.     

Stable Water Use Efficiency of Tibetan Alpine Meadows in Past Half Century: Evidence from Wool δ13C Values

Understanding the influences of climatic changes on water use efficiency (WUE) of Tibetan alpine meadows is important for predicting their long-term net primary productivity (NPP) because they are considered very sensitive to climate change. Here, we collected wool materials produced from 1962 to 2010 and investigated the long-term WUE of an alpine meadow in Tibet on basis of the carbon isotope values of vegetation (δ ¹³C_veg). The values of δ ¹³C_veg decreased by 1.34‰ during 1962–2010, similar to changes in δ ¹³C values of atmospheric CO₂. Carbon isotope discrimination was highly variable and no trend was apparent in the past half century. Intrinsic water use efficiency (W _i) increased by 18 μmol·mol^–1 (approximately 23.5%) during 1962–2010 because the increase in the intercellular CO₂ concentration (46 μmol·mol^–1) was less than that in the atmospheric CO₂ concentration (C _a, 73 μmol·mol^–1). In addition, W _i increased significantly with increasing growing season temperature and C _a. However, effective water use efficiency (W _e) remained relatively stable, because of increasing vapor pressure deficit. C _a, precipitation, and growing season temperature collectively explained 45% of the variation of W _e. Our findings indicate that the W _e of alpine meadows in the Tibetan Plateau remained relatively stable by physiological adjustment to elevated C _a and growing season temperature. These findings improve our understanding and the capacity to predict NPP of these ecosystems under global change scenarios.     

Pyrrole-imidazole hairpin polyamides with high affinity at 5'-CGCG-3' DNA sequence; influence of cytosine methylation on binding

To investigate the binding of 5′–CpG–3′ sequences by small molecules, two pyrrole (Py)–imidazole (Im) hairpin polyamides, PyImPyIm–γ–PyImPyIm–β–Dp (1) and PyIm–β–Im–γ–PyIm–β–Im–β–Dp (2), which recognize the sequence 5′–CGCG–3′, were synthesized. The binding affinities of the 5′–CGCG–3′ sequence to the Py–Im hairpin polyamides were measured by surface plasmon resonance (SPR) analysis. SPR data revealed that dissociation equilibrium constants (K_d) of polyamides 1 and 2 were 1.1 (± 0.3) × 10^–6 M and 1.7 (± 0.4) × 10^–8 M, respectively. Polyamide 2 possesses great binding affinity for this sequence, 65-fold higher than polyamide 1. Moreover, when all cytosines in 5′–CpGpCpG–3′ were replaced with 5-methylcytosines (^mCs), the K_d value of polyamide 2 increased to 5.8 (± 0.7) × 10^–9 (M), which indicated about 3-fold higher binding than the unmethylated 5′–CGCG–3′ sequence. These results suggest that polyamide 2 would be suitable to target CpG-rich sequences in the genome.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Higher Urinary Bisphenol A Concentration Is Associated with Unexplained Recurrent Miscarriage Risk: Evidence from a Case-Control Study in Eastern China

BackgroundEvidence about the association between Bisphenol A (BPA) and the risk of recurrent miscarriage (RM) in human being is still limited.ObjectiveWe evaluated the association of urinary BPA concentrations with RM in human being.MethodsA hospital-based 1:2 matched case-control study on RM was carried out in Suzhou and Kunshan in Jiangsu Province in China between August 2008 and November 2011. Total urinary BPA concentrations in 264 eligible urine samples (102 RM patients and 162 controls) were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The Wilcoxon test and conditional logistic regression were used to estimate the differences between the groups and odds ratios (OR) with 95% confidence intervals (CI), respectively.ResultsThe median ± IQR (interquartile range) (P₇₅-P₂₅) values of non-creatinine-adjusted total urinary BPA levels in the RM patients and the controls were 1.66±3.69ng/ml and 0.58±1.07ng/ml, respectively (0.98±2.67μg/g Cr (creatinine) and 0.40±0.77μg/g Cr. The adjusted BPA level was significantly higher in the RM patients than in the controls (Wilcoxon test, Z = 4.476, P<0.001). Higher level of urinary BPA was significantly associated with an increased risk of RM (P-trend <0.001). Compared to the groups with urinary BPA levels less than 0.16μg/g Cr, the women with levels of 0.40–0.93μg/g Cr and 0.93μg/g Cr or above had a significantly higher risk of RM (OR = 3.91, 95%CI: 1.23–12.45 and OR = 9.34, 95%CI: 3.06–28.44) that persisted after adjusting for confounding factors. The time from recently RM date to recruitment does not significantly influence the urinary BPA level (P = 0.090).ConclusionExposure to BPA may be associated with RM risk.     

Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (k_cat) for alginate of 0.60 ± 0.02 s⁻¹, 3.7 ± 0.3 s⁻¹, 4.5 ± 0.5 s⁻¹, and 7.1 ± 0.2 s⁻¹, respectively. The K_m values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds between β-d-mannuronate and α-l-guluronate and subunits; AlyD and AlyE were found to principally cleave the α-1,4 bonds involving α-l-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers.