Application of high-performance liquid chromatography of plasma fatty acids as their phenacyl esters to evaluate splanchnic and renal fatty acid balance in vivo

Plasma fatty acids from renal and hepatic veins, and arterialized hand vein obtained in 20 subjects before and after insulin infusion were separated by reversed-phase high-performance liquid chromatography following phenacyl esterification. Separation and quantification over the range 1.0–100 nmol per injection of nine fatty acids was achieved within 60 min using [²H₃₁]palmitic acid as internal standard. Analytical recoveries were greater than 90% and the intra- and inter-assay coefficients of variation were less than 2.5 and 4.0%, respectively. Following insulin infusion, net splanchnic uptake of total fatty acids decreased from 3.0±0.3 to 1.0±0.1 μmol/kg min (p<0.01), whereas net renal balance remained neutral (−0.04±0.04 vs. −0.06±0.03 μmol/kg min, p=N.S.). Individual fatty acid balance varied from a low of 0.012±0.005 (myristic acid) to a high of 0.95±0.08 (oleic acid) μmol/kg min across the splanchnic tissues and from 0.005±0.002 (stearic acid) to 0.21±0.1 (oleic acid) μmol/kg min across the kidney. There is a substantial diversity in changes in plasma concentration and regional balance of individual fatty acid during short-term fasting and hyperinsulinemia. This method is simple, accurate, and can be applied to assess individual fatty acid metabolism in vivo.     

Sensitive determination of xylenes in whole blood by capillary gas chromatography with cryogenic trapping

A new and sensitive method for measurement of o-, m- and p-xylenes in human whole blood by capillary gas chromatography (GC) with cryogenic trapping is presented. After heating 0.5 ml of whole blood and 0.5 ml of distilled water containing the xylenes and aniline (internal standard, I.S.) in a 4.0-ml vial at 100°C for 30 min, 2 ml of the headspace vapor was drawn into a glass syringe. All vapor was introduced through the GC port into an AT-Wax middle-bore capillary column in the splitless mode at an oven temperature of 5°C to trap the entire analytes, and the oven temperature was then programmed up to 180°C. The present conditions gave sharp peaks for xylenes and aniline (I.S.), and low background noises for whole blood samples; the peaks of p- and m-xylenes showed about 90% separation with the AT-Wax column. As much as 41.0–46.3% of xylenes, which had been spiked to whole blood could be recovered. The calibration curves showed linearity in the range of 0.1–0.5 μg/0.5 ml of whole blood. The detection limit was estimated to be about 10 ng/0.5 ml. The coefficients of intra-day and inter-day variations for xylenes were not greater than 9.38%. The data for actual detection of xylenes in post-mortem blood of self-ignition suicide cases by the present method were also presented.     

Liver Freezing Response of the Freeze-Tolerant Wood Frog, Rana sylvatica, in the Presence and Absence of Glucose. II. Mathematical Modeling

The “two-step” low-temperature microscopy (equilibrium and dynamic) freezing methods and a differential scanning calorimetry (DSC) technique were used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing, in Part I of this study. In this study, the experimentally determined dynamic water transport data are curve fit to a model of water transport using a standard Krogh cylinder geometry (Model 1) to predict the biophysical parameters of water transport: L_pg = 1.76 μm/min-atm and E_Lp = 75.5 kcal/mol for control liver cells and L_pg[cpa] = 1.18 μm/min-atm and E_Lp[cpa] = 69.0 kcal/mol for liver cells equilibrated with 0.4 M glucose. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5°C/min but do so when cooled at 2°C/min. Cells also retained twice as much intracellular fluid in the presence of 0.4 M glucose than in control tissue when cooled at 5°C/min. The ability of R. sylvatica liver cells to retain water during fast cooling (≥5°C/min) appears to be primarily due to its liver tissue architecture and not to a dramatically lower permeability to water, in comparison to mammalian (rat) liver cells which do dehydrate completely when cooled at 5°C/min. A modified Krogh model (Model 2) was constructed to account for the cell–cell contact in frog liver architecture. Using the same biophysical permeability parameters obtained with Model 1, the modified Krogh model (Model 2) is used in this study to qualitatively explain the experimentally measured water retention in some cells during freezing on the basis of different volumetric responses by cells directly adjacent to vascular space versus cells at least one cell removed from the vascular space. However, at much slower cooling rates (1–2°C/h) experienced by the frog in nature, the deciding factor in water retention is the presence of glucose and the maintenance of a sufficiently high subzero temperature (≥−8°C).     

Seasonal differences in activity of perch (Perca flavescens) gill Na/K ATPase

We measured Na⁺/K⁺ ATPase activity in homogenates of gill tissue prepared from field caught, winter and summer acclimatized yellow perch, Perca flavescens. Water temperatures were 2–4°C in winter and 19–22°C in summer. Na⁺/K⁺ ATPase activity was measured at 8, 17, 25, and 37°C. V_max values for winter fish increased from 0.48±0.07 μmol P mg⁻¹ protein h⁻¹ at 8°C to 7.21±0.79 μmol P mg⁻¹ protein h⁻¹ at 37°C. In summer fish it ranged from 0.46±0.08 (8°C) to 3.86±0.50 (37°C) μmol P mg⁻¹ protein h⁻¹. The K_m for ATP and for Na⁺ at 8°C was ≈1.6 and 10 mM, respectively and did not vary significantly with assay temperature in homogenates from summer fish. The activation energy for Na⁺/K⁺ ATPase from summer fish was 10 309 (μmol P mg⁻¹ h⁻¹) K⁻¹. In winter fish, the K_m for ATP and Na⁺ increased from 0.59±0.08 mM and 9.56±1.18 mM at 8°C to 1.49±0.11 and 17.88±2.64 mM at 17°C. The K_m values for ATP and Na did not vary from 17 to 37°C. A single activation energy could not be calculated for Na/K ATPase from winter fish. The observed differences in enzyme activities and affinities could be due to seasonal changes in membrane lipids, differences in the amount of enzyme, or changes in isozyme expression.     

Mechanism and Mechanism-Based Inactivation of 4-Hydroxyphenylpyruvate Dioxygenase

Six substrate analogs of 4-hydroxyphenylpyruvate, specifically pentafluorophenylpyruvate, 4-hydroxytetrafluorophenylpyruvate,2-thienylpyruvate, 3-thienylpyruvate, thiophenol oxalate, and p-thiocresoloxalate were synthesized and their interactions with porcine liver 4-hydroxyphenylpyruvate dioxygenase investigated. Both pentafluorophenylpyruvate and thiophenol oxalate are competitive inhibitors of the enzyme with K_I values of 14 and 150 μM, respectively, but p-thiocresol oxalate has no effect on the enzymic activity. The other three substrate analogs are both substrates and mechanism-based inactivators of the enzyme with the following kinetic characteristics (compound, K_m, V_max, k_inact, K′, partition ratio) at pH 6.0, 37°C, and an air atmosphere: 4-hydroxytetrafluorophenylpyruvate, 50 μM, 1.9 mkat/kg, 1.5/min, 70 μM 4.2; 2-thienylpyruvate, 500 μM, 7.8 mkat/kg, 0.6/min, 400 μM, 41; 3-thienylpymvate, 250 μM, 2 9 mkat/kg, 0.6/min, 300 μM, 22. When inactivated, the dioxygenase was found to contain per mole of active enzyme, 0.78 mol of label from 3-thienyl-3[³H]pyruvate and 0.85 mol of label from 4-hydroxytetrafluorophenyl-3 [³H]pyruvate. The product formed from the enzyme-catalyzed oxidation of 3-thienylpyruvate was determined to be 3-carboxymethyl-3-thiolene-2-one. The implication of these results to the mechanism of the dioxygenase is considered,     

Sensitive high-performance liquid chromatographic determination with fluorescence detection of phenol and chlorophenols with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a labeling reagent

A sensitive HPLC method for the determination of phenol and chlorophenols was developed. The fluorescence labeling reaction of phenols with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) was completed in 30 min at 60°C. The separation of DIB-derivatives of five representative phenols, i.e., phenol, o-, p-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, was achieved within 35 min with an ODS column using isocratic elution. The detection limits of these DIB derivatives at a signal-to-noise ratio (S/N) of 3 were in the range of 0.024 to 0.08 μM (0.12–0.45 pmol/20 μl injection). Twelve kinds of DIB derivatives with phenols containing mono-, di-, tri-, tetra- and penta-chlorophenol were also well separated within 208 min by changing the elution conditions. The derivatives were stable for at least for 24 h when they were placed at room temperature in the dark. The proposed method was applied to the assay of human urine samples and free and total phenol were determined. The relative standard deviations (RSDs) of the proposed method for within and between-day assay were <7.0% and <14.2%, respectively. The average concentrations of free and total phenol found in urine (n=6) were 4.3±2.5 and 29.5±14.0 μM, respectively.     

Growth characteristics of flagellated cells of Phaeocystis pouchetii revealed by diel changes in cellular DNA content

The present study reports on effects of different light:dark periods, light intensities, N:P ratios and temperature on the specific growth rate of flagellated cells of Phaeocystis pouchetii in culture. The specific growth rate was estimated by diel changes in cellular DNA content. The cellular DNA content and cell cycle of flagellated cells of P. pouchetii are shown, and the importance of light:dark period in cell division is demonstrated. Diel patterns of the cellular DNA content showed that cell division was confined to the dark period. The cells dealt with more than one division per day by rapid divisions shortly after each other.The specific growth rates (μ_DNA) based on the DNA cell cycle model were in close agreement with specific growth rates (μ_Cell) determined from cell counts. The temperature affected the specific growth rates (multiple regression, p < 0.01) and were higher at 5 °C (μ ≤ 2.2 d⁻¹) than at 10 °C (μ ≤1.6 d⁻¹). Increasing the light:dark period from 12:12 h to 20:4 h affected the specific growth rate of P. pouchetii at the lower temperature tested (5 °C) (multiple regression, p < 0.01), resulting in higher specific growth rates than at 10 °C. At 10 °C, the effect of light:dark period was severely reduced. Neither light nor nutrients could compensate the reduction in specific growth rates caused by elevated temperature. The specific growth rates was not affected by the N:P ratios tested (multiple regression, p = 0.21). The experiments strongly suggest that the flagellated cells have a great growth potential and could play a dominating role in northern areas at increased day length.     

Effects of temperature, salinity and irradiance on the growth of the red tide dinoflagellate Gyrodinium instriatum Freudenthal et Lee

The effects of temperature, salinity and irradiance on the growth of the red tide dinoflagellate Gyrodinium instriatum Freudenthal et Lee were examined in the laboratory. Exposed to 45 different combinations of temperature (10–30 °C) and salinity (0–40) under saturating irradiance, G. instriatum exhibited its maximum growth rate of 0.7 divisions/day at a combination of 25 °C and a salinity of 30. Optimum growth rates (>0.5 divisions/day) were observed at temperatures ranging from 20 to 30 °C and at salinities from 10 to 35. The organism could not grow at ≤10 °C. In addition, G. instriatum burst at a salinity of 0 at all temperatures, but grew at a salinity of 5 at temperatures between 20 and 25 °C. It is noteworthy that G. instriatum is a euryhaline organism that can live under extremely low salinity. Factorial analysis revealed that the contributions of temperature and salinity to its growth of the organism were almost equal. The irradiance at the light compensation point (I₀) was 10.6 μmol/(m² s) and the saturated irradiance for growth (I_s) was 70 μmol/(m² s), which was lower than I_s for several other harmful dinoflagellates (90–110 μmol/(m² s)).     

Lactate Oxidation for the Detection of Mitochondrial Dysfunction in Human Skin Fibroblasts

To screen fibroblasts for defects in lactate/pyruvate oxidation, cells were grown to confluence in 25-cm² flasks, rinsed, and incubated in glucose-free media containing 25 μM L-lactate and 0.1 μCi [D,L-1-¹⁴C]lactate. Lactate oxidation was measured as the amount of lactate oxidized in nmol of ¹⁴CO₂ generated /mg protein/min. Fibroblasts from patients with mitochondrial or peroxisomal disorders had decreased lactate oxidation compared to the control (CON): CON, 1.9 ± 0.13 nmol/mg/min; neonatal adrenoleukodystrophy (NALD), 0.45 ± 0.01 (P < 0.001); rhizomelic chondrodysplasia punctata (RCDP), 0.13 ± 0.002 (P < 0.001); mitochondrial defect of unknown etiology (MIT), 0.77 ± 0.003 (P <0.001); pyruvate dehydrogenase (PDH) deficiency, 0.98 ± 0.02 (P < 0.001). This method is useful for screening fibroblasts for defects in lactate oxidation in patients with mitochondrial or peroxisomal disorders. Confirmation of the site of the defect may then be investigated with specific assays, e.g., PDH, in cellular homogenates: CON, 0.93 ± 0.02 nmol/mg/min; NALD, 0.55 ± 0.02; RCDP, 0.44 ± 0.02; MIT, 0.53 ± 0.03; PDH deficiency, 0.19 ± 0.02.     

Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography—mass spectrometry using their N-labelled analogs

For the quantification of nitrite and nitrate, the stable metabolites of -arginine-derived nitric oxide (NO) in human urine and plasma, we developed a gas chromatographic—mass spectrometric (GC—MS) method in which [¹⁵N]nitrite and [¹⁵N]nitrate were used as internal standards. Endogenous nitrite and [¹⁵N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [¹⁵N]nitrate they were reduced to nitrite and [¹⁵N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-μl aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [¹⁵N]nitrite ([¹⁵N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects (n = 12) excreted into urine 0.49 ± 0.25 of nitrite and 109.5 ± 61.7 of nitrate (mean ± S.D., μmol/mmol creatinine) with a mean 24-h output of 5.7 μmol for nitrite and 1226 μmol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean ± S.D., μmol/l) 3.6 ± 0.8 and 68 ± 17, respectively.     

Membrane Permeability Characteristics of Metaphase II Mouse Oocytes at Various Temperatures in the Presence of Me2SO

In this study, the hydraulic conductivity (L_p), Me₂SO permeability ( _Me2SO), and the reflection coefficients (ς) and their activation energies were determined for Metaphase II (MII) mouse oocytes by exposing them to 1.5 M Me₂SO at temperatures of 30, 20, 10, 3, 0, and −3°C. These data were then used to calculate the intracellular concentration of Me₂SO at given temperatures. Individual oocytes were immobilized using a holding pipette in 5 μl of an isosmotic PBS solution and perfused with precooled or prewarmed 1.5 M Me₂SO solutions. Oocyte images were video recorded. The cell volume changes were calculated from the measurement of the diameter of the oocytes, assuming a spherical shape. The initial volume of the oocytes in the isoosmotic solution was considered 100%, and relative changes in the volume of the oocytes after exposure to the Me₂SO were plotted against time. Mean (means ± SEM) L_pvalues in the presence of Me₂SO ( ^Me₂SO_p) at 30, 20, 10, 3, 0, and −3°C were determined to be 1.07 ± 0.03, 0.40 ± 0.02, 0.18 ± 0.01, 7.60 × 10⁻²± 0.60 × 10⁻², 5.29 × 10⁻²± 0.40 × 10⁻², and 3.69 × 10⁻²± 0.30 × 10⁻²μm/min/atm, respectively. The _Me2SOvalues were 3.69 × 10⁻³± 0.3 × 10⁻³, 1.07 × 10⁻³± 0.1 × 10⁻³, 2.75 × 10⁻⁴± 0.15 × 10⁻⁴, 7.83 × 10⁻⁵± 0.50 × 10⁻⁵, 5.24 × 10⁻⁵± 0.50 × 10⁻⁵, and 3.69 × 10⁻⁵± 0.40 × 10⁻⁵cm/min, respectively. The ς values were 0.70 ± 0.03, 0.77 ± 0.04, 0.81 ± 0.06, 0.91 ± 0.05, 0.97 ± 0.03, and 1 ± 0.04, respectively. The estimated activation energies (E_a) for ^Me₂SO_p, _Me2SO, and ς were 16.39, 23.24, and −1.75 Kcal/mol, respectively. These data may provide the fundamental basis for the development of more optimal cryopreservation protocols for MII mouse oocytes.     

Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6–8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (<5 μg/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent K_m of 60 μM and V_max of 20 μmol·min⁻¹·mg⁻¹, corresponding values with mixed histones were 12 μM and 1.2 μmol·min⁻¹·mg⁻¹. With both protein substrates the apparent K_m for ATP was 11 μM. Concentrations of Mg²⁺ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7–2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated ³²P_i from [γ-³²P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.     

Determination of pyrazinamide and its main metabolites in rat urine by high-performance liquid chromatography

A new high-performance liquid chromatograhic procedure for simultaneous determination of pyrazinamide (PZA) and its three metabolites 5-hydroxypyrazinamide (5-OH-PZA), pyrazinoic acid (PA), and 5-hydroxypyrazinoic acid (5-OH-PA), in rat urine was developed. 5-OH-PZA and 5-OH-PA standards were obtained by enzymatic synthesis (xanthine oxidase) and checked by HPLC and GC–MS. Chromatographic separation was achieved in 0.01 M KH₂PO₄ (pH 5.2), circulating at 0.9 ml/min, on a C₁₈ silica column, at 22°C. The limits of detection were 300 μg/l for PZA, 125 μg/l for PA, 90 μg/l for 5-OH-PZA and 70 μg/l for 5-OH-PA. Good linearity (r²>0.99) was observed within the calibration ranges studied: 0.375–7.50 mg/l for PZA, 0.416–3.33 mg/l for PA, 0.830–6.64 mg/l for 5-OH-PZA and 2.83–22.6 mg/l for 5-OHPA. Accuracy was always lower than ±10.8%. Precision was in the range 0.33–5.7%. The method will constitute a useful tool for studies on the influence of drug interactions in tuberculosis treatment.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

Physical and stability characteristics of Burkholderia cepacia lipase encapsulated in κ-carrageenan

In this work, a novel approach for lipase immobilization was exploited. Lipase from Burkholderia cepacia was encapsulated into κ-carrageenan by co-extrusion method to form a liquid core capsule. The diameter of the encapsulated lipase was found to be in the range of 1.3–1.8 mm with an average membrane thickness of 200 μm and 5% coefficient of variance. The encapsulation efficiency was 42.6% and 97% moisture content respectively. The encapsulated lipase was stable between pH 6 and 9 and temperature until 50 °C. The encapsulated lipase was stable until disintegration of the carrier when stored at 27 °C and retained 72.3% of its original activity after 6 cycles of hydrolysis of p-NPP. The encapsulated lipase was stable in various organic solvents including methanol, ethanol, iso-propanol, n-hexane and n-heptane. Kinetic parameters K_m and V_max were found to be 0.22 mM and 0.06 μmol/min for free lipase and 0.25 mM and 0.05 μmol/min for encapsulated lipase respectively.     

Fast liquid chromatographic determination of urinary trans,trans-muconic acid

trans,trans-Muconic acid (1,3-butadiene-1,4-dicarboxylic acid, MA), a minor urinary metabolite of benzene exposure, was determined, after clean-up by solid-phase anion-exchange chromatography, by reversed-phase HPLC on a C₁₈ column (5 × 0.46 cm I.D., 3 μm particle size), using formic acid-tetrahydrofuran-water (14:17:969) as mobile phase and UV detection at 263 nm. The recovery of MA from spiked urine was > 95% in the 50–500 μg/l range; the quantification limit was 6 μg/l; day-to-day precision, at 300 μg/l, C.V. = 9.2%; the run time was less than 10 min. Urinary MA excretion was measured in two spot urine samples of 131 benzene environmentally exposed subjects: midday values obtained in non-smokers (mean±S.D.=77±54 μg/l, N = 82) were statistically different from those of smoerks (169±85 μg/l, N = 30) (P<0.0001); each group showed a statistically significant increase between MA excretion in midday over morning samples. Moreover, in subjects grouped according to tobacco-smoke exposure level, median values of MA were positively associated with and increased with daily smoking habits.     

Gas chromatographic—mass spectrometric assay to measure urinary N-methylhistamine excretion in man

An assay has been developed for N^τ-methylhistamine, a major metabolite of the autocoid histamine, based on gas chromatography—electron-capture negative-ion chemical ionisation mass spectrometry. N^τ-Methylhistamine was extracted from urine by cation-exchange chromatography and converted to its di-(3,5-bistrifluoromethylbenzoyl) derivative. The latter has good chromatographic properties and gives a negative-ion mass spectrum with the molecular ion (M, m/z 605) as base peak. A commercially available trideuterated analogue of N^τ-methylhistamine was used as internal standard. Basal urinary excretion of N^τ-methylhistamine in five normal subjects was found to be 0.21 ± 0.05 μmol/h (289 ± 74 μmol/mol of creatinine). This value was not significantly altered in these subjects following the infusion of a sub-pharmacological dose of histamine. In eight atopic volunteers, basal urinary excretion of N^τ-methyl-histamine was also not significantly changed following challenge with inhaled allergen.     

Effects of a synthetic prostaglandin analogue, cloprostenol, on the corpus luteum of the guinea pig

The effects of a synthetic prostaglandin analogue, cloprostenol, on luteal function in a guinea pig were studied. At a dose of 250μg, cloprostenol administered I-P on day 9 of the oestrous cycle caused a reduction in the length of the oestrous cycle from 17.4±s.d. 0.9 to 14.5±1.1 days (p<0.01). Lower doses were ineffective, and post-treatment cycles were not different in length from pre-treatment cycles. Cloprostenol also caused a dose-dependent reduction in luteal weight, which fell from 3.52±0.82 to 1.82±0.4mg (<0.01) 48 h after administation of a 250μg dose on day 9. Plasma progesterone, measured by radioimmunoassay, was reduced from 4.67±0.59 to 2.69±0.66 ng ml⁻¹(p<0.01) 48 h after administration of 250μg cloprostenol on day 9. 250μg cloprostenol also reduced blood flow per corpus luteum, measured by ⁸⁵Sr-labelled 15μm microspheres, both at 3 h (20.20±10.36 to 9.40±4.2μ1 min⁻¹; p0.05) and at 48 h 18.47±8.27 to 5.23±1.90μl min⁻¹; p<0.01) after administration on day 9. No adverse side-effects were observed at any dose level of cloprostenol used. It was concluded that cloprostenol is a useful experimental luteolysin in the guinea pig.     

Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile

An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent K_m and V_max values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min⁻¹ mg⁻¹, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose.