microM Min Research Articles

Intravital Imaging of IL-1β Production in Skin

IL-1 is a prototypic inflammatory cytokine that has pathogenic roles in various skin disorders. Although Langerhans cells (LCs) have been reported to express IL-1beta mRNA upon application of contact sensitizers, it remains unclear whether other cell types produce IL-1beta in skin. Thus, we sought to directly identify IL-1beta-producing cells in living animals by construction of transgenic mice expressing DsRed fluorescence protein gene under the control of IL-1beta promoter. Little DsRed fluorescence signal was detected in skin under steady-state conditions. Striking increases in DsRed signal were observed after topical application of a contact sensitizer, oxazolone, which also induced markedly elevated IL-1beta mRNA and protein expression. DsRed signal was expressed primarily by CD45(+)/CD11b(+) myeloid leukocytes in both epidermal and dermal compartments and was detected only in small fractions of epidermal LCs. Interestingly, DsRed(+) cells emerged preferentially as clusters around hair follicles. Intravital confocal imaging experiments revealed highly motile potentials of DsRed(+) cells-they constantly crawled around hair follicles via amoeba-like movements with a mean velocity of 1.0+/-0.4 microm min(-1) (epidermis) or 2.7+/-1.4 microm min(-1) (dermis). The newly developed in vivo imaging system represents a useful tool for studying spatial regulation of IL-1beta production in skin.

Speed of signal transfer in the chloroplast accumulation response

Chloroplast photorelocation movement is important for plants to perform efficient photosynthesis. Phototropins were identified as blue-light receptors for chloroplast movement in Arabidopsis thaliana and in the fern Adiantum capillus-veneris, whereas neochrome functions as a dual red/blue light receptor in the latter. However, the signal transduction pathways involved in chloroplast movement remain to be clarified. To investigate the kinetic properties of signalling from these photoreceptors to the chloroplasts, we deduced the speed of signal transfer using Adiantum capillus-veneris gametophytes. When a region of dark-adapted gametophyte cells was subjected to microbeam irradiation, chloroplasts moved towards the irradiated area even in subsequent darkness. We therefore recorded the movement and calculated the speeds of signal transfer by time-lapse imaging. Movement speeds under red or blue light were similar, e.g., about 1.0 microm min(-1) in prothallial cells. However, speeds varied according to cell polarity in protonemal cells. The speed of signal transfer from the protonemal apex to the base was approximately 0.7 microm min(-1), but roughly 2.3 microm min(-1) in the opposite direction. The speed of signal transfer in Arabidopsis thaliana mesophyll cells was approximately 0.8 microm min(-1) by comparison. Surprisingly, chloroplasts located farthest away from the microbeam were found to move faster than those in close proximity to the site of irradiation both in Adiantum capillus-veneris and A. thaliana.

An Alkaline Serine-Proteinase from a Bacterium Isolated from Bat Feces: Purification and Characterization

An alkaline serine-proteinase from Bacillus sp. PN51 isolated from bat feces collected in Phang Nga, Thailand, was purified and characterized. The molecular mass was estimated to be 35.0 kDa. The N-terminal 25 amino acid sequence was about 70% identical with that of Natrialba magadii halolysin-like extracellular serine protease. The enzyme showed the highest proteinase activity at 60 degrees C at pH 10.0. The activity was strongly inhibited by PMSF and chymostatin. The proteinase activity was not affected by the presence of 2% urea, 2% H(2)O(2), 12% SDS, 15% triton X-100, or 15% tween 80. The proteinase preferred Met, Leu, Phe, and Tyr residues at the P(1) position, in descending order. The k(cat), K(m) and k(cat)/K(m) values for Z-Val-Lys-Met-MCA were 16.8+/-0.14 min(-1), 5.1+/-0.28 microM, and 3.3+/-0.28 microM(-1) min(-1) respectively. This is the first report of an alkaline serine-proteinase with extremely high stability against detergents such as SDS.

Reliability of a pretransplant i.v. BU test dose performed 2 weeks before myeloablative FluBu conditioning regimen

A pretransplant test dose of i.v. BU was previously used in pediatric patients undergoing a reduced-intensity allogeneic hematopoietic SCT (HSCT). Here, we used a BU test dose in 23 adult patients who were not pancytopenic and underwent a myeloablative allogeneic HSCT prepared with fludarabine and i.v. BU (FluBU). Pharmacokinetics (PK) of BU were calculated after a test dose (0.8 mg/kg) was performed 2 weeks before transplant. Targeted BU area under the curve (AUC) range was 4800-5200 microM min. The mean BU dose calculated after the test dose was 3.5+/-0.5 mg/kg. To validate the test dose, PK studies were repeated in 17 patients after the first dose of BU during the conditioning regimen. An AUC below the therapeutic value of 4000 microM min was observed in 23% of the patients receiving a wt-based dose and in 0% of patients whose dose was calculated on the basis of the test dose (P=0.03). In patients who had a test dose, a significant correlation (P<0.0001) between the first and subsequent doses of BU during the conditioning regimen was observed. Our findings may allow more centers to pursue transplant strategies with targeted BU by overcoming the time limitation for PK studies during the conditioning regimen.

Cellular Biophysics During Freezing of Rat and Mouse Sperm Predicts Post-thaw Motility1

Though cryopreservation of mouse sperm yields good survival and motility after thawing, cryopreservation of rat sperm remains a challenge. This study was designed to evaluate the biophysics (membrane permeability) of rat in comparison to mouse to better understand the cooling rate response that contributes to cryopreservation success or failure in these two sperm types. In order to extract subzero membrane hydraulic permeability in the presence of ice, a differential scanning calorimeter (DSC) method was used. By analyzing rat and mouse sperm frozen at 5 degrees C/min and 20 degrees C/min, heat release signatures characteristic of each sperm type were obtained and correlated to cellular dehydration. The dehydration response was then fit to a model of cellular water transport (dehydration) by adjusting cell-specific biophysical (membrane hydraulic permeability) parameters L(pg) and E(Lp). A "combined fit" (to 5 degrees C/min and 20 degrees C/min data) for rat sperm in Biggers-Whitten-Whittingham media yielded L(pg) = 0.007 microm min(-1) atm(-1) and E(Lp) = 17.8 kcal/mol, and in egg yolk cryopreservation media yielded L(pg) = 0.005 microm min(-1) atm(-1) and E(Lp) = 14.3 kcal/mol. These parameters, especially the activation energy, were found to be lower than previously published parameters for mouse sperm. In addition, the biophysical responses in mouse and rat sperm were shown to depend on the constituents of the cryopreservation media, in particular egg yolk and glycerol. Using these parameters, optimal cooling rates for cryopreservation were predicted for each sperm based on a criteria of 5%-15% normalized cell water at -30 degrees C during freezing in cryopreservation media. These predicted rates range from 53 degrees C/min to 70 degrees C/min and from 28 degrees C/min to 36 degrees C/min in rat and mouse, respectively. These predictions were validated by comparison to experimentally determined cryopreservation outcomes, in this case based on motility. Maximum motility was obtained with freezing rates between 50 degrees C/min and 80 degrees C/min for rat and at 20 degrees C/min with a sharp drop at 50 degrees C/min for mouse. In summary, DSC experiments on mouse and rat sperm yielded a difference in membrane permeability parameters in the two sperm types that, when implemented in a biophysical model of water transport, reasonably predict different optimal cooling rate outcomes for each sperm after cryopreservation.

Direct visualisation and kinetic analysis of normal and nemaline myopathy actin polymerisation using total internal reflection microscopy

Actin filaments were formed by elongation of pre-formed nuclei (short crosslinked actin-HMM complexes) that were attached to a microscope cover glass. By using TIRF illumination we could see actin filaments at high contrast despite the presence of 150 nM TRITC-phalloidin in the solution. Actin filaments showed rapid bending and translational movements due to Brownian motion but the presence of the methylcellulose polymer network constrained lateral movement away from the surface. Both the length and the number of filaments increased with time. Some filaments did not change length at all and some filaments joined up end-to-end (annealing). We did not see any decrease in filament length or filament breakage. For quantitative analysis of polymerisation time course we measured the contour length of all the filaments in a frame at a series of time points and also tracked the length of individual filaments over time. Elongation rate was the same measured by both methods (0.23 microm/min at 0.1 microM actin) and was up to 10 times faster than previously published measurements. The annealed filament population reached 30% of the total after 40 min. Polymerisation rate increased linearly with actin concentration. K(on) was 2.07 microm min(-1) microM(-1) (equivalent to 34.5 monomers s(-1) microM(-1)) and critical concentration was less than 20 nM. This technique was used to study polymerisation of a mutant actin (D286G) from a transgenic mouse model. D286G actin elongated at a 40% lower rate than non-transgenic actin.

Age-dependent pharmacokinetic profile of single daily dose i.v. busulfan in children undergoing reduced-intensity conditioning stem cell transplant

We studied the pharmacokinetic (PK) profile of single daily dose i.v. BU in children who underwent reduced-intensity conditioning (RIC) transplantation. A cohort of 19 patients < or =4 years of age (group 1) and 33 patients >4 years (group 2) was studied. Patients received a BU test dose for PK studies, followed by two treatment doses adjusted to target an area under the curve (AUC) of 4000 microM min per day. Patients in group 1 attained a lower AUC as compared to group 2 (3568 vs 4035 microM min). In group 1, 67% patients and in group 2, 84% patients achieved AUC within the targeted range. Stable donor chimerism was achieved in 56% patients in group 1 and 79% in group 2. Eight patients required a second transplantation because of graft failure. Because of the concern that a low AUC adversely affected outcomes, a second cohort of 23 patients followed a modified protocol with a targeted AUC of 5000 microM min. A higher AUC was attained (4825 microM min). Stable donor chimerism was achieved in 91% of patients. Our results show that RIC regimens using two single daily doses of i.v. BU are effective in children, but a targeted AUC of 5000 microM min is recommended.

Enzymatic activity of surface-immobilized horseradish peroxidase confined to micrometer- to nanometer-scale structures in nanocapillary array membranes

Horseradish peroxidase (HRP) was immobilized on the planar surfaces and inside the cylindrical nanopores of nanocapillary array membranes (NCAMs) to study how the enzyme-catalyzed oxidation of a fluorigenic substrate, Amplex Red (AR), to fluorescent resorufin by hydrogen peroxide is influenced by confinement. Because AR was also found to be converted to resorufin photolytically at high laser fluences, a modified laser-induced fluorescence protocol was developed to characterize the enzyme-catalyzed reaction in the absence of interference from the photolytic reaction. Surface-immobilized HRP was studied in two environments: bound to the surface of a microfluidic channel, and bound to the interior of cylindrical nanopores in NCAMs connecting crossed microfluidic channels. HRP was immobilized through reaction of solvent-accessible primary amines with the epoxy group of the methyl methacrylate-glycidyl methacrylate copolymer synthesized in either planar or annular geometries to construct the test structures for enzymatic activity. HRP immobilized on planar surfaces shows high activity (approximately 10 microM min(-1)) meaning that the copolymer membrane exhibits good potential for immobilizing the enzyme, especially since active structures are obtained in a one-step reaction. HRP was also immobilized inside nanopores via physisorption. Enzymatic reactions inside the nanopores were characterized and compared to finite element simulations of a modified Eley-Rideal mechanism to bracket the value of the overall rate constant for the confined enzyme. Reaction velocities were estimated to be approximately 10-fold higher in the nanopores than for the same enzyme bound to a planar microfluidic surface.

Plasma and cerebrospinal fluid pharmacokinetics of tasidotin (ILX-651) and its metabolites in non-human primates

To evaluate the plasma and cerebrospinal fluid (CSF) pharmacokinetics and CSF penetration of tasidotin and metabolites in a nonhuman primate model. Tasidotin 0.75 mg/kg was administered intravenously. The plasma and CSF concentrations of tasidotin and its metabolites were determined. Pharmacokinetic parameters were estimated using model-independent and model-dependent methods. The mean (+/-SD) CSF:plasma AUC ratio for tasidotin was 1.1 +/- 0.4. For tasidotin, tasidotin-C-carboxylate and desprolyl-tasidotin-C-carboxylate the plasma AUCs (mean +/- SD) were 30 +/- 10, 54 +/- 19 and 12 +/- 2 microM min, and apparent plasma half-lives were 27 +/- 4, 229 +/- 73 and 100 +/- 29 min. The plasma clearance of tasidotin was 44 +/- 14 ml/min/kg. The CSF AUC and half-life of tasidotin was 28 +/- 10 microM min and 96 +/- 40 min. The model-dependent plasma clearance was 35 ml/min/kg for tasidotin and 2 ml/min/kg for tasidotin-C-carboxylate. Tasidotin penetrates into the CSF well and further evaluation of its activity in the treatment of central nervous system malignancies should be considered.

Ligand Binding to the Fe(III)-Protoporphyrin IX Complex of Phosphodiesterase from Escherichia coli (Ec DOS) Markedly Enhances Catalysis of Cyclic di-GMP: Roles of Met95, Arg97, and Phe113 of the Putative Heme Distal Side in Catalytic Regulation and Ligand Binding

Phosphodiesterase (Ec DOS) from Escherichia coli is a gas-sensor enzyme in which binding of gas molecules, such as O(2), CO, and NO, to the Fe(II)-protoporphyrin IX complex in the sensor domain stimulates phosphodiesterase activity toward cyclic-di-GMP. In this study, we report that external axial ligands, such as cyanide or imidazole, bind to Fe(III)-protoporphyrin IX in the sensor domain and induce a 10- to 11-fold increase (from 8.1 up to 86 min(-1)) in catalysis, which is more substantial than that (6.3 to 7.2-fold) observed for other gas-stimulated Fe(II) heme-bound enzymes. Catalytic activity (50 min(-1)) of the heme-free mutant, H77A, was comparable to that of the ligand-stimulated enzymes. Accordingly, we propose that the heme at the sensor domain inhibits catalysis and that ligand binding to the heme iron complex releases this catalytic suppression. Furthermore, mutations of Met95, Arg97, and Phe113 at the putative heme distal side suppressed the ligand effects on catalysis. The rate constants (19,000 x 10(-5) microM(-1)min(-1)) for cyanide binding to the M95A and M95L mutants of the full-length enzyme were 633-fold higher than that to wild-type Ec DOS (30 x 10(-5) microM(-1)min(-1)). The absorption spectrum of the F113Y mutant suggests that the Tyr O(-) group directly coordinates to the Fe(III) complex and that the cyanide binding rate to the mutant is very slow, compared with those of the wild-type and other mutant proteins. We observed a similar trend in the binding behavior of imidazole to full-length mutant enzymes. Therefore, while Met95 and Phe113 are not direct axial ligands for the Fe(III) complex, catalytic, spectroscopic, and ligand binding evidence suggests that these residues are located in the vicinity of the heme.

Kinetics of Whole Serum and Prepurified IgG Digestion by Pepsin for F(ab')2 Manufacture

An alternative route for the production of polyclonal F(ab')(2) fragments that might be adopted for the facile preparation of antivenoms is assessed in this work. The method involves the digestion of whole serum by free pepsin, which results in reduction of the number of processing steps commonly in use, because it avoids the initial purification of IgG's prior to their proteolytic cleavage by the enzyme. Digestion kinetics of whole serum and caprylic acid prepurified IgG using free pepsin were monitored with SDS-PAGE followed by densitometric analysis and antigen binding activity assay of the digested samples. It was observed that with equal units of pepsin activity, caprylic acid prepurified IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was significantly greater in the latter case. The estimated first-order digestion rate parameters were 11.8 and 4.42 microM min(-)(1) for pure IgG and whole serum, respectively. The K(m) value obtained for whole serum digestion was 33 microM and that for pure IgG digestion was 43.5 microM. Calibration with undigested whole serum and pure IgG samples of known concentrations was performed using SDS-PAGE followed by image analysis. A linear relationship was observed between the protein concentration and the respective band intensity within the range of concentrations investigated (0.63-31.2 microM IgG concentration). This technique proved to be relatively rapid, reproducible, and more precise than size-exclusion chromatography as a result of its F(ab')(2)/IgG resolving power. Staining and destaining protocols were reproduced in terms of staining and destaining times, volumes added, and compositions. Furthermore, all digestion experiments were performed in duplicate sets to monitor the extent of variation of the digestion kinetic parameters measured by this method. The results obtained from this technique confirm and quantify previous observations that pepsin digestion of whole serum is slower and easier to control than digestion of pure IgG and results in higher recovery of antigenic binding activity.

Cold atmospheric plasma disinfection of cut fruit surfaces contaminated with migrating microorganisms.

The efficacy of cold atmospheric gas plasmas against Escherichia coli type 1, Saccharomyces cerevisiae, Gluconobacter liquefaciens, and Listeria monocytogenes Scott A was examined on inoculated membrane filters and inoculated fruit surfaces. Inoculated samples were exposed to a cold atmospheric plasma plume generated by an AC voltage of 8 kV at 30 kHz. The cold atmospheric plasma used in this study was very efficient in reducing the microbial load on the surfaces of filter membranes. However, its efficacy was markedly reduced for microorganisms on the cut surfaces. This lack of effect was not the result of quenching of reactive plasma species responsible for microbial inactivation but principally the result of the migration of microorganisms from the exterior of the fruit tissue to its interior. The velocity of migration through melon tissues was estimated to be around 300 microm min(-1) for E. coli and S. cerevisiae and through mango tissues to be 75 to 150 microm min(-1). These data can serve as operational targets for optimizing the performance of gas plasma inactivation processes. The current capabilities of cold atmospheric plasmas are reviewed and ways to improve their bactericidal efficacy are identified and discussed. Considerable scope exists to enhance significantly the efficacy of cold atmospheric plasmas for decontaminating fresh cut fruits.

A 52-kDa Leucyl Aminopeptidase from Treponema denticola Is a Cysteinylglycinase That Mediates the Second Step of Glutathione Metabolism

The metabolism of glutathione by the periodontal pathogen Treponema denticola produces hydrogen sulfide, which may play a role in the host tissue destruction seen in periodontitis. H2S production in this organism has been proposed to occur via a three enzyme pathway, gamma-glutamyltransferase, cysteinylglycinase (CGase), and cystalysin. In this study, we describe the purification and characterization of T. denticola CGase. Standard approaches were used to purify a 52-kDa CGase activity from T. denticola, and high pressure liquid chromatography electrospray ionization tandem mass spectrometry analysis of this molecule showed that it matches the amino acid sequence of a predicted 52-kDa protein in the T. denticola genome data base. A recombinant version of this protein was overexpressed in and purified from Escherichia coli and shown to catalyze the hydrolysis of cysteinylglycine (Cys-Gly) with the same kinetics as the native protein. Surprisingly, because sequence homology indicates that this protein is a member of a family of metalloproteases called M17 leucine aminopeptidases, the preferred substrate for the T. denticola protein is Cys-Gly (k cat/Km of 8.2 microm(-1) min(-1)) not l-Leu-p-NA (k cat/Km of 1.1 microm(-1) min(-1)). The activity of CGase for Cys-Gly is optimum at pH 7.3 and is enhanced by Mn2+, Co2+, or Mg2+ but not by Zn2+ or Ca2+. Importantly, in combination with the two other previously purified T. denticola enzymes, gamma-glutamyltransferase and cystalysin, CGase mediates the in vitro degradation of glutathione into the expected end products, including H2S. These results prove that T. denticola contains the entire three-step pathway to produce H2S from glutathione, which may be important for pathogenesis.

Valacyclovir and acyclovir pharmacokinetics in immunocompromised children

Valacyclovir, an orally administered pro-drug of acyclovir, is utilized in the therapy of herpes simplex and herpes zoster infections. Little data regarding the pharmacokinetics, safety and tolerability are available for pediatric patients. This report describes acyclovir pharmacokinetics following valacyclovir administration in immunocompromised pediatric patients, compares pharmacokinetic parameters following oral valacyclovir and IV acyclovir, and provides a limited assessment of efficacy in the setting of active herpes zoster infection. A total of 37 immunocompromised children were enrolled on one of two studies. Pharmacokinetic data are available for 32 patients following valacyclovir (15 mg/kg) administration, 11 of whom also had pharmacokinetic sampling following IV acyclovir administration. Three patients received valacyclovir as treatment for herpes zoster infections. Mean (+/-SD) C(max) values for acyclovir following oral valacyclovir were 18.8 +/- 7 microM with a total exposure of 4,106 +/- 1,519 microM min. The mean bioavailability of acyclovir from valacyclovir was 64%. Grade 1 nausea and emesis, which occurred in five patients was the only valacyclovir-related toxicity. Two of the three patients treated for herpes zoster had complete scabbing of lesions by day 9. Valacyclovir (15 mg/kg) was well tolerated in pediatric patients and demonstrated excellent bioavailability. Consideration should be given to the use of oral valacyclovir for the treatment of herpes zoster in clinically stable pediatric oncology patients.

Surface Behavior and Peptide−Lipid Interactions of the Cyclic Neuropeptide Melanin Concentrating Hormone

The kinetics and the thermodynamics of melanin concentrating hormone (MCH) adsorption, penetration, and mixing with membrane components are reported. MCH behaved as a surface active peptide, forming stable monolayers at a lipid-free air-water interface, with an equilibrium spreading pressure, a collapse pressure, and a minimal molecular area of 11 mN/m, 13 mN/m, and 140 A (2), respectively. Additional peptide interfacial stabilization was achieved in the presence of lipids, as evidenced by the expansion observed at pi > pi sp in monolayers containing premixtures of MCH with zwitterionic or charged lipids. The MCH-monolayer association and dissociation rate constants were 9.52 x 10 (-4) microM (-1) min (-1) and 8.83 x 10 (-4) min (-1), respectively. The binding of MCH to the dpPC-water interface had a K d = 930 nM at 10 mN/m. MCH penetration in lipid monolayers occurred even up to pi cutoff = 29-32 mN/m. The interaction stability, binding orientation, and miscibility of MCH in monolayers depended on the lipid type, the MCH molar fraction in the mixture, and the molecular packing of the monolayer. This predicted its heterogeneous distribution between different self-separated membrane domains. Our results demonstrated the ability of MCH to incorporate itself into biomembranes and supports the possibility that MCH affects the activity of mechanosensitive membrane proteins through mechanisms unrelated with binding to specific receptors.

Laboratory evolution of P450 BM3 for mediated electron transfer yielding an activity-improved and reductase-independent variant

One of the main obstacles in employing P450 monooxygenases for preparative chemical syntheses in cell-free systems is their requirement for cofactors such as NAD(P)H. In order to engineer P450 BM3 from Bacillus megaterium for cost-effective process conditions in vitro, a validated medium throughput screening system based on cheap Zn dust as an electron source and Cobalt(III)sepulchrate (Co(III)sep) as a mediator was reported. In the current study, the alternative cofactor system Zn/Co(III)sep was used in a directed evolution experiment to improve the Co(III)sep-mediated electron transfer to P450 BM3. A variant, carrying five mutations (R47F F87A V281G M354S D363H, Table I), P450 BM3 M5 was identified and characterized with respect to its kinetic parameters. P450 BM3 M5 achieved for mediated electron transfer a 2-fold higher k(cat) value and a 3-fold higher catalytic efficiency compared with the starting point mutant P450 BM3 F87A (k(cat): 62 min(-1) compared with 28 min(-1); k(cat)/K(m): 62 microM(-1)min(-1) compared to 19 microM(-1)min(-1)). For obtaining first insights on electron transfer contributions, three reductase-deficient variants were generated. The reductase-deficient mutant of P450 BMP M5 exhibited a catalytic efficiency of 69% and a k(cat) value of 89% of the values obtained for P450 BM3 M5.

FRET‐Based Direct and Continuous Monitoring of Human Fucosyltransferases Activity: An Efficient synthesis of Versatile GDP‐L‐Fucose Derivatives from Abundant d‐Galactose

We have developed a facile and versatile protocol for the continuous monitoring of human fucosyltransferases activity by using fluorescence energy resonance transfer (FRET), and have explored the feasibility of its use in an inhibitor screening assay. A convenient sugar nucleotide with a fluorogenic probe, 6-deoxy-6-N-(2-naphalene-2-yl-acetamide)-beta-L-galactopyranos-1-yl-guanosine 5'-diphosphate disodium salt (1), was efficiently synthesized from naturally abundant D-galactopyranose via a key intermediate, 6-azide-1,2,3,4-tetra-O-benzoyl-6-deoxy-beta-L-galactopyranose (10). It was demonstrated that the combined use of the glycosyl donor 1 and a dansylated acceptor substrate, sialyl-alpha2,3-LacNAc derivative (2) allowed us to carry out highly sensitive, direct, and continuous in vitro monitoring of the generation of sialyl Lewis X (SLe x), which is catalyzed by human alpha-1,3-fucosyltransferase VI (FUT-VI). A kinetic analysis revealed that compound 1 was an excellent donor substrate (KM=0.94 microM and Vmax=0.14 microM min(-1)) for detecting human FUT-VI activity. To the best of our knowledge, this synthetic fluorogenic probe is the most sensitive and selective donor substrate for FUT-VI among all of the known GDP-Fuc analogues, including the parent GDP-Fuc. When a dansylated asparagine-linked glycopeptide 20, which is derived from egg yolk was employed as an alternate acceptor substrate, a FRET-based assay with compound 1 could be used to directly monitor the alpha1,6-fucosylation at the reducing terminal GlcNAc residue by human FUT-VIII (KM=175 microM and Vmax=0.06 microM/ min); this indicates that the present method might become a general protocol for the characterization of various mammalian fucosyltransferases in the presence of designated fluorogenic acceptor substrates. The present protocol revealed that compound 23, which was obtained by a 1,3-dipolar cycloaddition between the disodium salt 16 and 1-ethynyl-naphthalene exhibits highly potent inhibitory effects against the FUT-VI-mediated sialyl Lewis X synthesis (IC50=5.4 microM).

Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell's osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V(b)) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and -38.1+/-1.2%. The permeability of the oocytes to water (L(p)) was determined to be 3.8+/-0.4 x 10(-2), 5.7+/-0.8 x 10(-2), and 13.2+/-1.3 x 10(-2) microm min(-1)atm(-1), when measured at temperatures of 5, 10 and 20 degrees C. The respective EG permeability values (P(s)) were 9.5+/-0.1 x 10(-5), 14.6+/-1.2 x 10(-5), and 41.7+/-2.4 x 10(-5) cm min(-1). The activation energies for L(p) and P(s) were determined to be 14.5 and 17.5 kcal mol(-1), respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 degrees C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.

Permeability of human oocytes to ethylene glycol and their survival and spindle configurations after slow cooling cryopreservation

To develop novel cryopreservation methods, we estimated the permeability coefficients Lp (hydraulic conductivity) and P(EG) (cryoprotectant permeability) of mature human oocytes after exposure to ethylene glycol (EG) and tested the efficiency of a multi-step slow cooling protocol based on this cryoprotectant. Oocytes were perfused with 1.5 mol/l EG for 10 min. Oocyte volume at each time point was calculated and normalized to the original volume. Slow cooling was conducted by exposing oocytes to increasing EG concentrations (0.5, 1.0 and 1.5 mol/l n = 155) or 1.5 mol/l of propane-1,2-diol (PrOH) n = 102. Oocytes which survived cryopreservation n = 80 and fresh oocytes n = 73 were prepared for confocal microscopy analysis of the meiotic spindle. During EG exposure, oocytes underwent an abrupt 50% volume reduction. Complete recovery of the initial volume was not observed. From the values of a best fit plot, the coefficients Lp = 0.82 +/- 0.29 microm min(-1) atm(-1) (mean +/- SD) and P(EG) 0.10 +/- 0.01 microm s(-1) were generated. Survival rates after freezing with EG were lower than with PrOH (51.6 versus 71.5%, respectively, P < 0.05). The frequencies of normal spindle configuration were lower in frozen EG and frozen PrOH oocytes compared with fresh oocytes (53.8, 50.9 and 66.7%, respectively, P < 0.05). The oocyte plasmalemma possesses limited permeability to EG and EG exposure causes considerable osmotic stress. However, post-thaw rates of survival and normal meiotic spindle organization may be preserved by protocols which are designed in order to minimize osmotic stress.

Two trypsin isoforms from the intestine of the grass carp (Ctenopharyngodon idellus)

Two trypsin isoforms (GT-A and GT-B) from the grass carp (Ctenopharyngodon idellus) intestine were isolated and purified. SDS-PAGE electrophoresis showed that GT-A and GT-B had relative molecular masses of 30,740 and 26,400, respectively. Enzyme activity was inhibited by three organic trypsin inhibitors but not by EDTA. They had optimal pH of 8.0 and 8.5, and optimal temperatures of 38.5 and 44.0 degrees C, respectively, when hydrolyzing N-benzoyl-L: -arginine ethyl ester.HCl (BAEE). They lost 95.8 and 93.7% of their activities, respectively, after heating for 20 min at 65 degrees C. Their thermal denaturation temperatures, respectively, were 66.3 and 67.3 degrees C. GT-A has a K(m) value of 21.2 microM and a V(max) of 2.0 x 10(3) min(-1), and GT-B has a K(m) value of 31.7 microM and a V(max) of 3.3 x 10(3) min(-1). Their physiological efficiencies were 94.3 and 105.3 microM(-1) min(-1), respectively. The Arrhenius activation energies of GT-A and GT-B were 4.16 and 4.38 kcal/mol, respectively. The activities of GT-A and GT-B were not activated by Ca(2+), but their thermostability was improved in the presence of Ca(2+). Enzyme activity was reduced in presence of Zn(2+), Cu(2+), Hg(2+) and Al(3+). Thermal stabilities of GT-A and GT-B were intermediate between Arctic and tropical fish species, and consistent with the wide range of water temperatures to which grass carp are exposed in most provinces of China.