F S Wang — Page 4 | PubMed Health

PubMedDate not supplied

Immobilization of whole-cell penicillin G acylase by entrapping within polymethacrylamide beads.

Escherichia coli ATCC 11105 containing the periplasmic penicillin G acylase was entrapped within a copolymer of methacrylamide and N,N'-methylenebisacrylamide. A solution of monomer that was made up from methacrylamide and N,N'-methylenebisacrylamide dissolved in buffer was mixed with lyophilized cells and ammonium persulfate. This suspension was then pumped drop by drop into in soybean oil supplemented with 0.06% (v/v) 3-(dimethylamino)-propionitril. During submerging in the oil phase, the droplets were hardened and induced to polymerize within the droplets. Particles with a volume ranging from 0.013-0.017 mL per bead containing a biomass concentration up to 38.0 g/L were prepared. The optimal condition for the deacylation of penicillin G to 6-aminopencillanic acid (6-APA) catalyzed by the immobilized whole-cell penicillin G acylase was found to be 45 degrees C and pH 8.0. Product inhibition of this enzyme by 6-APA could be eliminated by controlling pH value at 8 during the course of penicillin G hydrolysis using a pH-stat. Conversion determined by the pH-stat method were 0.3% higher than that by p-dimethylaminobenzaldehyde method. Cell concentration in the matrix was found to be an important factor influencing the maximum velocity and the specific activity retained in the matrix. A kinetic model, in which the mass transfer resistances as a result of external film mass transfer and pore diffusion were assumed to be negligible, could properly describe the hydrolysis of penicillin G by the cells entrapped within the polymethacylamide beads.

Cell Count↗

PubMedDate not supplied

Relative susceptibility of cytoskeleton-associated and soluble neurofilament subunits to aluminum exposure in intact cells. A possible mechanism for reduction of neurofilament axonal transport during aluminum neurotoxicity.

Previous studies have demonstrated the appearance of phosphorylated neurofilament (NF) subunits within perikaryal cytoskeletons following aluminum exposure. In order to examine the mechanisms leading to this altered distribution of NF subunits, we carried out biochemical analyses of NF subunits in Triton-insoluble and -soluble fractions derived from aluminum-treated NB2a/d1 cells. In addition to increases in the Triton-insoluble cytoskeleton, increases in all three NF subunits were also detected within the Triton-soluble fraction of aluminum-treated cells. To address the nature of this increase in Triton-soluble subunits, aluminum-treated and untreated cultures were harvested in the absence of Triton and fractionated by established procedures to yield fractions greatly enriched for perikarya and neurites, respectively. Each of these subcellular fractions was then subjected to further homogenization in the presence of 1% Triton and centrifugation to yield Triton-insoluble cytoskeletons and Triton-soluble material derived from perikarya and axonal neurites, respectively. Resulting Triton-soluble fractions were "clarified" by high-speed centrifugation to eliminate oligomeric assemblies or soluble neurofilaments. Immunoblot analysis demonstrated quantitative recovery of the aluminum-induced increase in Triton-soluble NF subunits in the perikaryal fraction. Additional aluminum-treated and untreated cultures were pulse-chase radiolabeled with [35S]methionine and fractionated into Triton-insoluble and soluble fractions from isolated perikarya and axonal neurites. Autoradiographic analysis of immunoprecipitated NF subunits revealed that aluminum treatment delayed the translocation of newly synthesized subunits into neurites and resulted in the accumulation of radiolabeled subunits within the Triton-soluble fraction of perikarya. These findings suggest that aluminum may exert a relatively greater effect on NF subunits that have not yet undergone axonal transport and/or incorporation into Triton-insoluble structures vs those that have already deposited into axons. This possibility was supported by the observation that a higher concentration of aluminum was required to alter the electrophoretic migration of in vitro reassembled neurofilaments vs that required for unassembled NF subunits. These findings provide possible mechanisms for the accumulation of NF subunits in perikarya during aluminum intoxication.

Aluminum↗