Purification and some properties of tyrosinase from aspergillus flavipes 56003

A method for isolation and purification of tyrosinase from the fungus Aspergillus flavipes 56003 was developed. The method includes extraction with water, concentration on DEAE-cellulose, gel-filtration on Acrylex P-150, and ion-exchange chromatography on DEAE-Toyopearl 650M. The tyrosinase was purified to apparent homogeneity according polyacrylamide gel electrophoresis and ultracentrifugation. The tyrosinase is a 130-kD protein with pI 4.6. It contains two copper atoms. The Km and Vmax for tyrosine hydroxylation are 0.3 mM and 1300 &mgr;moles/min per mg at pH 6.8, and for dehydrogenation of 3,4-dihydroxyphenylalanine (DOPA) they are 5 mM and 16000 &mgr;moles/min per mg, respectively. Hydroxylation of monophenols has a characteristic lag period. The rate of tyrosine and DOPA oxidation is maximal at pH 6.0-6.8. The half-life of the enzyme at 50 degrees C is 40 min. The hydroxylase activity of the tyrosinase is more stable at neutral pH, whereas the dehydrogenase activity is more stable at acidic pH (4.0). The absorption spectrum of the enzyme has a maximum at 290 mn and a shoulder in the 320-400-nm region.     

Optimization of the medium and conditions for cultivatingErwinia sp., a producer of fumarase and aspartase

Mathematical methods of experimental design were used to determine the optimal concentrations of nutrient medium components, aeration conditions, and pH providing for the maximum biomass yields, as well as fumarase and aspartase activities, during submerged cultivation ofErwinia sp. The data showed that different concentrations of carbon source (molasses) and pH of the nutrient medium were required to reach the maximum yields of fumarase and aspartase. Calculations suggested that the combination of these optimized factors would result in 3.2-, 3.4-, and 3.8-fold increases in theErwinia sp. biomass, aspartase activity, and fumarase activity yields, respectively. The experimental data were consistent with these estimates to 80% accuracy.     

Multiplex Genotyping of Allelic Variants of Genes Involved in Metabolizing Antileukemic Drugs

A biochip, primer set, and genotyping protocol were developed to simultaneously address 16 single nucleotide polymorphisms in antileukemic drug metabolism genes, including TPMT, ITPA, MTHFR, SLCO1B1, SLC19A1, NR3C1, GRIA1, ASNS, MTRR, and ABCB1. The genotyping procedure included a one-round multiplex polymerase chain reaction (PCR) with simultaneous incorporation of a fluorescent label into the PCR product and subsequent hybridization on a biochip with immobilized probes. The method was used to test 65 DNA samples of leukemia patients. Fluorescence signal intensity ratios in pairs of wildtype and respective mutant sequence probes were analyzed for all polymorphic markers and demonstrated high accuracy of genotyping. The reliability of genotype determination using the biochip was confirmed by direct Sanger sequencing.     

Study of the Kinetics of Oxidation of Monophenols by Tyrosinase. The Effect of Reducers

Kinetics of oxidation of monophenols by tyrosinase from the fungus Aspergillus flavipes 56003 and the effect of Fe²⁺, serine, and ascorbic acid on this reaction were studied. The effectors were shown to accelerate the oxidation of monophenols, decreasing the lag-time of the reaction. It is assumed that the activation of the tyrosinase in the presence of Fe²⁺ is due to a direct reduction of the active site copper ions. Serine and ascorbic acid are supposed to affect the reaction of quinone transformation. The activation of the enzyme in the presence of Fe²⁺ suggests that the oxidation of monophenols is an autocatalytic process.     

Pathogenesis of diphtheria carrier state from the immunological point of view.

Results of a comparative investigation of diphtheria antitoxin and type-specific antibacterial antibodes in 264 carriers of diphtherial bacteria, 41 diphtheria patients and 263 non-infected subjects are presented. A high level of antitoxin did not prevent the development of toxigenic-strain carrier state. A basically similar immunological antibacterial response was observed in patients with manifest forms of diphtheria and in carriers of toxigenic strains; such a response could not as yet be detected in carriers of non-toxigenic strains. It has been suggested that the infectious process in the toxigenic-strain carrier state is due to factors of the virulence responsible for infectivity and invasiveness of the diphtherial microbe. The toxin plays no pathogenic role in carrier state.     

Solution Formulation Development of a VEGF Inhibitor for Intravitreal Injection

PF-00337210 is a potent, selective small molecule inhibitor of VEGFRs and has been under consideration for the treatment of age-related macular degeneration. An ophthalmic solution formulation intended for intravitreal injection was developed. This formulation was designed to maximize drug properties such that the formulation would precipitate upon injection into the vitreous for sustained delivery. As a parenteral formulation with additional constraints dictated by this specialized delivery route, multiple features were balanced in order to develop a successful formulation. Some of these considerations included low dosing volumes (≤0.1 mL), a limited repertoire of safe excipients for intravitreal injection, and the unique physical chemical properties of the drug. The aqueous solubility as a function of pH was characterized, buffer stressing studies to select the minimal amount of buffer were conducted, and both chemical and physical stability studies were executed. The selected formulation consisted of an isotonic solution comprised of PF-00337210 free base in a citrate-buffered vehicle containing NaCl for tonicity. The highest strength for regulatory toxicology studies was 60 mg/mL. The selected formulation exhibited sufficient chemical stability upon storage with no precipitation, and acceptable potency and recovery through an intravitreal dosing syringe. Formulation performance was simulated by precipitation experiments using extracted vitreous humor. In simulated injection experiments, PF-00337210 solutions reproducibly precipitated upon introduction to the vitreous so that a depot was formed. To our knowledge, this is the first time that a nonpolymeric in situ-forming depot formulation has been developed for intravitreal delivery, with the active ingredient as the precipitating agent.     

Characterization of cyclic AMP-regulated chloride conductance in the pigmented rabbit conjunctival epithelial cells

We have previously reported that the pigmented rabbit conjunctiva is a Cl- secreting tissue, subject to cAMP, Ca2+, and PKC modulation. The present study was conducted to characterize, at the cellular and molecular levels, cAMP-regulated Cl- channels in rabbit conjunctival epithelial cells. cAMP-inducible Cl- channel properties were evaluated by monitoring the whole-cell currents using patch clamp techniques. Results showed that 10 microM forskolin significantly stimulated a glibenclamide-inhibitable whole-cell conductance by approximately five-fold. Furthermore, reduction of the Cl- concentration in the bathing solution through partial substitution of NaCl with Na-isethionate resulted in a rightward shift of the reversal potential for both baseline and forskolin-stimulated whole-cell currents from 0 to values close to the theoretical Cl- reversal potential predicted by the Nernst equation. Western blot analysis with a monoclonal antibody recognizing the epitope in the C-terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) showed a positive band at its molecular weight, approximately 170 kD. Immunostaining under confocal microscopy revealed a CFTR specific signal in the apical sections of primary conjunctival epithelial cells. In addition, RT-PCR detection amplified a cDNA fragment 100% identical to the predicted portion of the cloned rabbit CFTR message. The stage is thus set for determining the extent of CFTR contribution to cAMP-regulated Cl- conductance in pigmented rabbit conjunctival epithelial cells.     

Pharmacological modulation of fluid secretion in the pigmented rabbit conjunctiva

We determined net fluid secretion rate across the pigmented rabbit conjunctiva in the presence and absence of pharmacological agents known to affect active Cl- secretion and Na+ absorption. Fluid flow across a freshly excised pigmented rabbit conjunctiva mounted between two Lucite half chambers was measured by a pair of capacitance probes in an enclosed cabinet maintained at 37 degrees C and a relative humidity of 70%. Fluid transport was also measured in the presence of compounds known to affect active Cl- secretion (cAMP, UTP, and ouabain), Na+ absorption (D-glucose), or under the Cl--free condition on both sides of the tissue. Net fluid secretion rate across the pigmented rabbit conjunctiva in the serosal-to-mucosal direction at baseline was 4.3+/-0.2 microl/hr/cm2 (mean +/- s.e.m.). Net fluid secretion rate was increased approximately two-fold by mucosally applied 1 mM 8-Br cAMP (8.4+/-0.4 microl/hr/cm2) and 10 microM UTP (9.8+/-0.6 microl/hr/cm2), but was abolished by either serosally applied 0.5 mM ouabain (0.3+/-0.1 microl/hr/cm2) or under the Cl--free conditions (0.06+/-0.04 microl/hr/cm2). Mucosal addition of 20 mM D-glucose decreased net fluid secretion rate to 1.0+/-0.5 microl/hr/cm2. In conclusion, the pigmented rabbit conjunctiva appears to secrete fluid secondary to active Cl- secretion. This net fluid secretion is subject to modulation by changes in active Cl- secretion rate and in mucosal fluid composition such as glucose concentration.     

Thermodynamic stoichiometry of Na+-coupled glutathione transport

Ambiguity exists with respect to mechanisms of glutathione (GSH) transport and the molecular identity of GSH transporters. Empirical and theoretical limitations have hindered functional and molecular characterizations. Published literature referring to the isolation and molecular identification of Na+-coupled GSH transporters that mediate the cellular uptake of GSH is highly debated. Whereas a number of functional and kinetic reports of this putative symport mechanism exist, the hypothetical transmembrane Na+-coupled GSH transporter protein or the genetic message encoding it has not been isolated. Theoretical thermodynamic calculations to support the concept of secondary active GSH transport and to rationalize accounts of physical-kinetic measurements describing Na+-coupled cellular GSH uptake were performed. The adequacy of requisite energy and stoichiometric conservation of the separate electrical and chemical components of a Na+ gradient in maintaining a high cellular accumulation gradient for GSH was examined through a purely phenomenological perspective. Dependent on the biological context, the energetic coupling between Na+ and GSH cotransport may occur at ratios from 1:1 to 3:1. Molecular identification of specific transporters responsible for cellular Na+-coupled GSH uptake will facilitate determination of their relative contribution to the overall plasma membrane resting potential. In tissues with a high GSH concentration relative to their extracellular milieu, particularly in pathologies of cystic fibrosis and dry eye syndromes, large energy coupling ratios in cotransport of Na+ and GSH may be expected. Na+-coupled GSH transport may play an important role in disease onset and (or) progression, or treatment modalities thereof.