Calculation of the molecular masses of two newly synthesized thermostable enzymes isolated from thermophilic microorganisms

Two thermostable enzymes synthesized by thermophilic microorganisms were isolated and purified. A thermostable ß-galactosidase was produced in a continuous fermentation process by Bacillus stearothermophilus TP 32 as an intracellular enzyme. After applying different concentration procedures the raw extract enzyme was prepurified on a Sephadex G-200 size exclusion column. The isolated ß-galactosidase fraction was then separated with HPLC on a TSK G 3000 SW size exclusion column to determine the molecular mass based on calibration curves of standard proteins. The other enzyme, a thermostable protease, was synthesized by Bacillus stearothermophilus TP 26 as an extracellular enzyme. After its concentration, the enzyme was purified on a classical size exclusion column (Sephacryl S-200) and on a HPLC size exclusion column (BIO-SIL TSK-250). The micropreparatively isolated fraction was separated again on this HPLC column to determine its molecular mass. The optimum temperature of both enzymes was approximately 75°C.     

山溪鲵的脊柱特征,兼议有尾目脊柱的划分

为探讨有尾目脊椎的划分,本文以小鲵科的山溪鲵(Batrachuperus pinchonii)为例,运用透明骨骼双色法对其脊柱的形态特征进行了观察,并对各部分椎骨特征进行详细描述和绘图.结果显示,山溪鲵的脊椎根据椎骨是否具前关节突、横突、肋骨、肋软骨和脉弓等形态特征可分5部分;同时结合小鲵科其他20种94号标本和蝾螈科6种27号标本的脊柱特征及文献资料,讨论了有尾目脊椎的划分,认为将有尾目脊柱划分为5部分(颈椎、躯椎、荐椎、尾荐椎和尾椎)的观点较将其划分为4部分(颈椎、躯椎、荐椎和尾椎)的观点更合理.     

Selective separation of human peripheral platelets,granulocytes and lymphocytes by surface affinity chromatography

Selective separation of human peripheral platelets, granulocytes and lymphocytes was investigated by column liquid chromatography using methoxyethoxymethyl (MEM) bonded-phase columns (25 × 0.9 cm I.D.). Isotonic solutions containing mono- and disaccharides, methyl-α-d-pyranosides and a physiological saline at pH 7.4 were used as the mobile phase. Granulocytes and lymphocytes were separated on the MEM-Cellulofine GH-25 column by elution with 0.3 M d-mannose solution. The isolation of platelets and lymphocytes from human leukocyte-rich plasma was performed with a MEM-Sephadex G25 column and elution with 0.27 M sucrose solution. On the same column platelets could also be collected selectively by elution with 0.31 M methyl-α-d-mannoside at the high recovery of 100%. The isolated cells were viable for more than 90%.     

Global distribution and diversity of marine Verrucomicrobia

Verrucomicrobia is a bacterial phylum that is commonly detected in soil, but little is known about the distribution and diversity of this phylum in the marine environment. To address this, we analyzed the marine microbial community composition in 506 samples from the International Census of Marine Microbes as well as 11 coastal samples taken from the California Current. These samples from both the water column and sediments covered a wide range of environmental conditions. Verrucomicrobia were present in 98% of the analyzed samples, and thus appeared nearly ubiquitous in the ocean. Based on the occurrence of amplified 16S ribosomal RNA sequences, Verrucomicrobia constituted on average 2% of the water column and 1.4% of the sediment bacterial communities. The diversity of Verrucomicrobia displayed a biogeography at multiple taxonomic levels and thus, specific lineages appeared to have clear habitat preference. We found that subdivision 1 and 4 generally dominated marine bacterial communities, whereas subdivision 2 was more frequent in low salinity waters. Within the subdivisions, Verrucomicrobia community composition were significantly different in the water column compared with sediment as well as within the water column along gradients of salinity, temperature, nitrate, depth and overall water column depth. Although we still know little about the ecophysiology of Verrucomicrobia lineages, the ubiquity of this phylum suggests that it may be important for the biogeochemical cycle of carbon in the ocean.     

Dynamic model of a nitrifying fixed bed column: Simulation of the biomass distribution of Nitrosomonas and Nitrobacter and of transient behaviour of the column

A dynamic model for a fixed bed nitrifying column with recirculation of the liquid and gas phases was developed. Liquid RTD experiments demonstrated that the liquid phase was perfectly mixed inside the column. Hete- rogeneity of biomass distribution on the solid phase (beads) was represented by an N-tanks in series model, and a back-mixing term was set to account for the well-mixed liquid phase throughout the column. In autotrophic conditions, competition for oxygen is the cause of the spatial segregation of the two species. Nitrosomonas is concentrated on beads at the bottom of the bed whereas Nitrobacter is more widely distributed. This is consistent with biomass distribution results reported by Cox et al. [17] in a nitrifying fixed bed column. Nitrification takes place at the bottom of the column, always in oxygen gas-liquid mass transfer limiting conditions. Nevertheless, considering the whole process, nitrification is complete (>98% of NH₃ oxidised) and there is no oxygen limitation (the outlet dissolved oxygen concentration is not limiting). The dynamic behaviour of the column, in conditions set up to avoid biofilm diffusion limitation, was simulated for different NH₃-load variations and oxygen shutdowns. The simulated behaviour of the column can be compared to results reported by Bazin et al. [16]. This confirms that the output transient nitrite peaks are higher when changes in the process conditions produce a rearrangement of biomass distribution in the fixed bed.     

Taxonomy of barkeria (Orchidaceae)

The genusBarkeria (Orchidaceae) consists of four species and eight subspecies (the treatment includes a key) of epiphytic plants endemic to Mexico and Central America.Barkeria is separated from the genusEpidendrum on the structure of the rostellum and column in the flower, plus shape of the pseudobulbs. The authors suggest thatBarkeria is most closely related toCaularthron based on the general shape of the flowers, widely spreading fleshy column wings, and structure of the rostellum.     

New internal standards for basic amino acid analyses

Eight derivatives of cysteine and penicillamine with 2- and 4-vinyl-pyridine, p-nitrobenzyl bromide, and p-nitrostyrene were evaluated as potential internal standards for the short and long (physiological) basic columns in amino acid analysis by ion-exchange chromatography. S-β-(4-pyridylethyl)-dl-penicillamine (4-PEP) was found to have an advantage over the previously proposed S-β-(4-pyridylethyl)-l-cysteine (4-PEC) since the elution position of 4-PEP on the short basic column is insensitive to minor changes in pH of the eluting citrate buffer. 4-PEP was found to be stable to acid hydrolysis as used for proteins and its recovery from protein hydrolysates was unaffected by the presence of starch during hydrolysis. However, an extra 14 min is required to elute 4-PEP on the short column.Of the eight compounds studied, six appcar suitable as internal standards on the physiological (long) column. These elute in widely differing positions between histidine and arginine, thus offering a choice of internal standards for special analysis on the basic long column.     

Purification of almond emulsin alpha-L-fucosidase I by affinity chromatography.

An affinity chromatographic method to purify α-l-fucosidase I from almond emulsin was developed. A derivative of lacto-N-fucopentaose II, ?-aminocaproyl-lacto-N-fucopentaosylamine, was coupled to sepharose 4B and packed in a column. By adopting this column for affinity chromatography, the enzyme was purified a hundredfold. The enzyme preparation was free from any other exoglycosidases which act on natural substrates.     

Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound ah-sepha-rose 4b

Heparinase and heparitinase were separated from an extract of Flavobacterium heparinum, induced with heparin by using column chromatography on hydroxylapatite. As the heparinase preparation contained chondroitinases B and C, chondroitinase B was removed by rechromatography on a hydroxylapatite column. Chondroitinase C was then eliminated by column chromatography on O-phosphono(“phospho”)-cellulose. The heparinase preparation thus obtained was free from sulfoamidase for 2-deoxy-2-sulfoamino-D-glucose (GlcN-2S), sulfatase for 2-amino-2-deoxy-6-O-sulfo D-glucose (GlcN-6S), as well as (δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin. The remaining sulfatase for 4-deoxy-α-L-thero-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) in the heparinase preparation was removed by affinity chromatography with dermatan sulfate-bound AH-Sepharose 4B coated with dermatan sulfate. The heparitinase preparation separated by column chromatography on hydroxyla patite was purified by affinity chromatography with heparin-bound AH-Sepharose 4B coated with heparin. Sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S) and δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin were removed by this chromatography. Sulfatase for 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) remaining in the heparitinase preparation was finally removed by column chromatography on hydroxylapatite. The recoveries of the purified preparations of heparinase and heparitinase were estimated to be 39 and 50%, respectively, from the crude enzyme fractions obtained by the first column chromatography on hydroxyl- patite. The purified heparinase and heparitinase were free from all enzymes that could degrade the sulfated unsaturated disaccharides produced from heparin with heparinase.     

Affinity chromatography without immobilized ligands; A new method for studying macromolecular interactions using high-performance liquid chromatography

A new method for studying macromolecular interactions was devised. The principle is based on affinity chromatography with a mobile zone of affinity ligand instead of a column with immobilized ligand. In this method, the difference in migration velocities between the moving zone of affinity ligand and a sample in a conventional gel-permeation column is utilized. The fast migrating zone (A zone), which is later injected into the column, and the slow migrating zone (B zone), which is injected beforehand, interfere with each other at the passing point in the column if A and B interact such as A + B ? AB. The zone interference deforms each elution profile of A and B, because the complex AB has a migration velocity different from the others. The elution profiles in zone-interference chromatography are calculated by computer simulation in the framework of the plate theory. The binding constant is calculated from the peak shift of elution volume of B in the zone-interference chromatogram. The interaction between single-strand DNA (A zone) and ribonuclease A (B zone) was studied.     

Fiber entrapment of naringinase from Penicillium sp. and application to fruit juice debittering

Naringinase from Penicillium sp. was immobilized on cellulose triacetate by the fiber entrapment method. Although the optimum pH (3.7) and optimum temperature (55°C) of the fiber-entrapped enzyme were similar to those of the native form, the immobilized enzyme had better heat stability. Kinetic studies showed that the immobilized enzyme had higher K_m values than its native form. When this immobilized naringinase was successively used in a column reactor for the hydrolysis of ρ-nitrophenyl-α-l-rhamnoside or naringin in a simulated fruit juice system or grapefruit juice, the enzyme column could be operated with satisfactory stability. In addition, when the natural grapefruit juice was recycled through the column reactor, no column blocking or filtering action of the catalyst bed was observed.     

Spectral Characterization of the Photoreducible b-Type Cytochrome of Dictyostelium discoideum

Irradiation of a soluble extract from broken cells of Dictyostelium discoideum causes the photoreduction of a b-type cytochrome. The cytochrome b can be separated from cytochrome c, which is also present in the extract, by column chromatography on Brushite, but the cytochrome b is no longer sensitive to light after separation on the column. Low temperature spectroscopy shows that reduced form of the photoreducible cytochrome b has a Soret band at 423 nm and a split α band with maxima at 558 and 551 nm similar to the b-type cytochrome in complex II of beef heart mitochondria.     

Analysis of thiohydantoins from amino acids by gas-liquid chromatography on short glass capillary columns

A method for separation of amino acid methyl or phenyl thiohydantoins by GLC on a short glass capillary column is described. Calculations of required parameters of the capillary column are presented. By the described methods, nineteen of twenty silylated methylthiohydantoins were separated in one run. The last one (histidine) can be identified on the same column by starting the analysis at a higher temperature. Cysteine and arginine were analysed as S-methylcysteine and ornithine, respectively.     

Flow velocity affects internal oxygen conditions in the seagrass Cymodocea nodosa

The internal oxygen status of seagrass tissues, which is believed to play an important role in events of seagrass die-off, is partly determined by the rates of gas exchange between leaves and water column. In this study, we examined whether water column flow velocity has an effect on gas exchange, and hence on internal oxygen partial pressures (pO₂) in the Mediterranean seagrass, Cymodocea nodosa. We measured the internal pO₂ in the horizontal rhizomes of C. nodosa in darkness at different mainstream flow velocities, combined with different levels of water column oxygen pO₂ using an experimental flume in the laboratory. Flow velocity clearly had an effect on the internal oxygen status. In stagnant, but fully aerated water the mean internal pO₂ was 6.9 kPa, corresponding to about 30% of air saturation. The internal pO₂ increased with increasing flow velocity reaching saturation of around 12.2 kPa (60% of air saturation) at flow velocities ≥7 cm s⁻¹. Flow had a relatively larger influence on internal pO₂ at lower water column oxygen concentrations. By extrapolating linear relationships between internal and water column pO₂ in this experimental setup, rhizomes would become anoxic at a water column oxygen pO₂ of 4–4.5 kPa (∼20% of air saturation) in flowing water, but already at 6.4 kPa (∼30% of air saturation) in stagnant water. Water flow may play an important role for seagrass performance and survival in areas with poor water column oxygen conditions and may, in general, be of importance for the distribution of submerged rooted plants.     

Effects of nonuniform column packing in analytical gel chromatography. II. Associating solutes

Effects of nonuniform column packing on boundary profiles for selfassociating systems have been investigated by computer simulation. Migration rate of each of the interconverting solute species changes along the column as a result of nonuniform packing, and the difference in velocity of monomer and n-mer is not constant as the sample moves down the column. A greater amount of overall axial dispersion results, as compared to the constant-column case. Procedures developed in this study can be applied to any experimentally measured column nonuniformity.     

Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (20×4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 mM phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35×2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100×2 mm I.D.) using 36% acetonitrile in 10 mM phosphate solution (pH 4.5) when the valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD≤2.3%) and accuracy (bias: ±2.0%) and speed (total analysis time 17 min). The response was linear (r²≥0.999) over the concentration range 10–1000 ng/ml.     

Author index for volume 84

A rapid method for hemoglobin chain recombination which gives a homogeneous product was developed. The method utilizes a small carboxymethylcellulose column as a medium for chain recombination and concentration of the hemoglobin. Equimolar amounts of p-hydroxymercuribenzoate derivatives of α- and β-chains were mixed with 300× molar excess of β-mercaptoethanol over the p-hydroxy mercuribenzoate groups. After 10 min of incubation in an ice bath, the mixture was adjusted to pH 5.85, and was loaded on a carboxymethylcellulose column. The column was washed with 10 mm phosphate buffer-1 mm Na₂EDTA-47 mm β-mercaptoethanol, pH 5.85 and then with 10 mm phosphate buffer, pH 5.85. The hemoglobin was eluted from the column by use of 15 mm K₂HPO₄. The hemoglobin was homogeneous on polyacrylamide gel electrophoresis and had a visible spectrum, electrophoretic mobility, and number of -SH groups comparable to those shown by control hemoglobin.     

Enantioselective determination of selfotel in human urine by high-performance liquid chromatography on a chiral stationary phase after derivatization with 9-fluorenylmethyl chloroformate

An analytical method for the enantioselective determination of selfotel in human urine has been developed and validated. The method is based on high-performance liquid chromatography and utilizes CGS 20005 (a selfotel analog) as the internal standard. Urine samples were derivatized in situ with o-phthalic dicarboxaldehyde–3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate (FMOC). Chromatographic separations of the FMOC derivatives of selfotel enantiomers and the internal standard were achieved using a column switching system consisting of an Inertsil ODS-2 column (75×4.6 mm I.D., 5 μm) and a Chiralcel OD-R column (250×4.6 mm I.D., 10 μm). The composition of the mobile phase was acetonitrile–0.1 M phosphate buffer, pH 2.50 (35:65) for the Inertsil ODS-2 column and acetonitrile–0.1 M phosphate buffer, pH 2.00 (35:65) for the Chiralcel OD-R column. The analytes were monitored using fluorescence detection at an excitation wavelength of 262 nm and an emission wavelength of 314 nm. The limit of quantification (LOQ) for this method is 0.25 μg/ml for each selfotel enantiomer. The method was successfully utilized to determine preliminary selfotel stereospecific pharmacokinetics.     

Effect of Soil Depth and Moisture on the Vertical Distribution of Steinernema riobrave (Nematoda: Steinernematidae)

The effect of soil moisture on the distribution of Steinernema riobrave in a sand column was determined. Larvae of Pectinophora gossypiella were used to detect S. riobrave infective juveniles (IJ) in each 2.5-cm section of 30-cm-long soil columns. Soil moisture was determined for each section and related to the numbers of nematodes recovered from infected insect baits. Infective juveniles of S. riobrave applied on the sand column surface showed some degree of positive geotaxis. IJ in soil columns with a consistent moisture gradient grouped in the upper 12.7 cm within a water potential range of ¯40 to ¯0.0055 MPa (2% to 14% moisture). Nematodes in sand columns that were gradually dehydrating moved down the soil column, aggregating on the 28th day between 15-23 cm in depth. Nematode redistribution over time allowed IJ to remain within a water potential range of ¯0.1 to ¯0.012 MPa (5.2% to 9.5% moisture).     

Electrical properties of the body wall of Hydra

If the hydrostatic pressure in the enteron of Hydra is made more than 2–4 mm of water greater than the outside pressure, the animal becomes distended, indicating that the normal enteron pressure is less than this. Positive enteron pressure attenuates the spontaneous, negative-going electrical spikes across the body wall, which are called contraction pulses (CP''s) because of their relation to column contraction. Pressure has little effect on the transepithelial resting potential. The low frequency electrical impedance of the column is nonlinear. The impedance tends to increase as the transepithelial potential is made more negative. The nonlinearity has both initial and delayed components. The DC impedance of the column near the resting potential averages 100 kohms (approximately 5 kohms-cm²). The phase between transepithelial potential and imposed sinusoidal current approaches -90° with increasing current frequency. Bode plots of the column impedance and the phase lag indicate that the column has three or more time constants. CP''s show several unusual features: (a) their amplitude and frequency are essentially independent of the transepithelial potential when the latter is altered by imposed current; (b) there is practically no change in column impedance during CP firing; (c) when the transepithelial potential is clamped at zero, CP''s continue to appear spontaneously as current spikes. These features are consistent with the hypothesis that the CP-generating membrane forms but a small fraction of the total transverse impedance of the column.