Retention of enzyme by electropolymerized film: a new approach in developing a hypoxanthine biosensor

An amperometric biosensor for hypoxanthine was constructed by forming a layer of crosslinked xanthine oxidase on a platinum electrode, followed by electropolymerization of a submonolayer film of resorcinol and para-diaminobenzene. The fabricated electrodes were evaluated for speed of response, sensitivity, and reusability. Optimal performance was obtained with enzyme-based electrodes sparsely covered with film which was formed by electropolymerization in less than 6 min. The resulting electrodes exhibited linear response to hypoxanthine in the. range 5-300 muM with a response time of 2 min. Application of the biosensor in monitoring hypoxanthine content of fish extracts yielded results which agreed well with spectrophotometric assays using soluble xanthine oxidase. The biosensor was stable for 60 days when stored at 4 degrees C in phosphate buffer and it could be used continuously for 6 h with over 50 assays.     

Inhibitors of Urokinase and Thrombin in Cultured Neural Cells

Recent studies have suggested important roles for certain proteases and protease inhibitors in the growth and development of the CNS. In the present studies, inhibitors of urokinase or thrombin in cultured neural cells and serum-free medium from the cells were identified by screening for components that formed sodium dodecyl sulfate-stable complexes with 125I-urokinase or 125I-thrombin. Rinsed glioblastoma possessed two components that complexed 125I-urokinase. One was type 1 plasminogen activator inhibitor (PAI-1), because the 125I-urokinase-containing complexes were immunoprecipitated with anti-PAI-1 antibodies. The other component formed complexes with 125I-urokinase that were not recognized by antibodies to PAI-1 or protease nexin-1 (PN-1). Its identity is unknown. In addition to these cell-bound components, the glioblastoma cells also secreted two inhibitors that formed complexes with 125I-urokinase; one was PAI-1, and the other was PN-1. The secreted PN-1 also formed complexes with 125I-thrombin. It was the only thrombin inhibitor detected in these studies. Human neuroblastoma cells did not contain components that formed detectable complexes with either 125I-urokinase or 125I-thrombin. However, human neuroblastoma cells did contain very low levels of PN-1 mRNA and PN-1 protein. Added PN-1 bound to the surface of both glioblastoma and neuroblastoma cells. This interaction accelerated the inhibition of thrombin by PN-1 and blocked the ability of PN-1 to form complexes with 125I-urokinase. Thus, cell-bound PN-1 was a specific thrombin inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity and regulation of manganese peroxidases from Phanerochaete chrysosporium.

Lignin and Mn peroxidases are two families of isozymes produced by the lignin-degrading fungus Phanerochaete chrysosporium under nutrient nitrogen or carbon limitation. We purified to homogeneity the three major Mn peroxidase isozymes, H3 (pI = 4.9), H4 (pI = 4.5), and H5 (pI = 4.2). Amino-terminal sequencing of these isozymes demonstrates that they are encoded by different genes. We also analyzed the regulation of these isozymes in carbon- and nitrogen-limited cultures and found not only that the lignin and Mn peroxidases are differentially regulated but also that differential regulation occurs within the Mn peroxidase isozyme family. The isozyme profile and the time at which each isozyme appears in secondary metabolism differ in both nitrogen- and carbon-limited cultures. Each isozyme also responded differently to the addition of a putative inducer, divalent Mn. The stability of the Mn peroxidases in carbon- and nitrogen-limited cultures was also characterized after cycloheximide addition. The Mn peroxidases are more stable in carbon-limited cultures than in nitrogen-limited cultures. They are also more stable than the lignin peroxidases. These data collectively suggest that the Mn peroxidase isozymes serve different functions in lignin biodegradation.     

Replication-defective mutants of herpes simplex virus (HSV) induce cellular immunity and protect against lethal HSV infection.

Live viruses and live virus vaccines induce cellular immunity more readily than do inactivated viruses or purified proteins, but the mechanism by which this process occurs is unknown. A trivial explanation would relate to the ability of live viruses to spread and infect more cells than can inactivated virus. We have used live but replication-defective mutants to investigate this question. Our studies indicate that the immune responses of mice to live virus differ greatly from the responses to inactivated virus even when the virus does not complete a replicative cycle. Further, these studies indicate that herpes simplex virus-specific T-cell responses can be generated by infection with replication-defective mutant viruses. These data indicate that the magnitude of the cellular immunity to herpes simplex virus may be proportional to the number or quantity of different viral gene products expressed by an immunizing virus.     

Purification and characterization of cytosolic 6-phosphogluconate dehydrogenase isozymes from maize

Cytosolic isozymes of 6-phosphogluconate dehydrogenase were purified from roots of maize (Zea mays L.). The final preparation contained two 55-kD proteins. Affinity-purified dehydrogenases from a maize line that is null for both cytosolic 6-phosphogluconate dehydrogenase isozymes (Pgd1-null, Pgd2-null) lacked the 55-kD proteins. The substrate kinetics of the purified enzyme were determined.     

A rapid method for counting cell nuclei using a particle sizer/counter

Summary We report here an improved method for nuclei counting utilizing Triton-X 100 to reduce the size of cell debris, thereby allowing the use of a particle sizer/counter. Furthermore, nuclei are completely released within 30 seconds, as compared to 1 hour using hypotonic solution. The method is accurate above 0.3 × 10⁶ cells/mL.     

Studies on the primary sequence requirements for PKC-alpha, -beta 1 and -gamma peptide substrates.

The substrate specificity of purified PKC-alpha, -beta and -gamma has been investigated. A series of synthetic peptides based upon the sequence surrounding serine-7 in glycogen synthase were generated and used to determine the basic residue requirements of these PKC isotypes. While PKC-alpha and -beta are indistinguishable in their phosphorylation of these peptides, PKC-gamma shows a distinct specificity profile for these synthetic substrates.     

Interaction between corticosteroid binding globulin and activated leukocytes in vitro

The interaction between human corticosteroid binding globulin and activated leukocytes is restricted to the granulocyte population, and is characterized by specific proteolytic cleavage of corticosteroid binding globulin which markedly reduces its steroid binding activity. A direct interaction between corticosteroid binding globulin and the activated cells appears to enhance this event, and does not involve cellular internalization of corticosteroid binding globulin or its proteolytic degradation products, which resemble those obtained after incubation of corticosteroid binding globulin with neutrophil elastase. These data suggest that corticosteroid binding globulin interacts with elastase on the surface of activated neutrophils, and may promote glucocorticoid delivery to these cells during inflammation.