Membrane enzyme reactor with simultaneous separation using electrophoresis

A membrane enzyme reactor with simultaneous separation was investigated. Enzymes, urease and aspartase, were immobilized by a porous polytetrafluoroethylene membrane. Electrical field was applied in the medium while the reaction was carried out. Products with electrical charge could be separated through the membrane from the reaction medium as they were formed. Reaction behavior was analyzed by a simple model considering both pore-migration and reaction in the skelton of the membrane. According to the analysis the inherent reaction rate of the immobilized enzymes decreases significantly. This is probably caused by the structural variation of enzymes. For the case of urease, the change of pH inside the membrane may also cause the decrease of the reaction rate. The model analysis showed that the enzyme content in the membrane and the residence time of the substrate in the membrane governed overall extent of reaction.List of Symbols e g (dm³)^–1 enzyme concentration in the membrane - L cm membrane thickness - K _m mM Michaelis constant - Rate mmol · min^–1 · g^–1 rate of product formation per unit weight of enzyme - S mM substrate concentration - S _in mM inlet substrate concentration - S _out mM outlet substrate concentration - u cm · min^–1 migration rate - V V voltage between the electrodes - V _m mmol · min^–1 · g^–1 maximum reaction rate - X conversion - z cm distance from the surface inside the membrane - void fraction of the porous membrane - tortuosity of the membrane - min space time     

Insensitivity of maximum expiratory flow to bronchodilation in normal dogs

We examined the effects of the inhaled parasympatholytic agent atropine and the sympathomimetic agent salbutamol on partitioned frictional pressure (Pfr) losses to the site of flow limitation (choke point, CP) in dogs to see how changes brought about by these agents would affect maximum expiratory flow (Vmax) and response to breathing 80% He-20% O2 (delta Vmax) in terms of wave-speed theory of flow limitation. In open-chest dogs, a Pitot-static tube was advanced down the right lower lobe to locate CP, to determine CP lateral and end-on pressures (PE), and to partition the airway into peripheral (alveoli to sublobar) and central (sublobar to CP) segments. Measurements were obtained at approximately 50% vital capacity. After inhalation, CP locations were unchanged with both bronchodilating agents. After atropine inhalation, Pfr central was decreased by one-half compared with base line. Despite the decrease in Pfr central, however, Vmax failed to increase after atropine because of altered bronchial area pressure (BAP) behavior at the CP site. After salbutamol inhalation, Pfr peripheral was reduced by about one-half compared with base line. However, Vmax failed to increase, because this reduction was too small to significantly increase the CP pressure head (i.e., PE). delta Vmax was also insensitive to these agents. Our results show mechanisms by which small changes in Pfr, as well as the complex interaction of changes in Pfr and BAP, may limit the use of Vmax in detecting bronchodilation at different airway sites.     

Cell-Cell Recognition during Fertilization in a Red Alga, Antithamnion sparsum (Ceramiaceae, Rhodophyta)

A gamete recognition mechanism in Antithamnion sparsum Tokidais proposed based on experiments using various lectins and carbohydrates.Spermatial binding to trichogynes is inhibited by pre-incubationof spermatia with concanavalin A (ConA) and/or L-fucose, whiletrichogyne receptors are blocked by the complementary carbohydrate-methyl D-mannose and/or the lectin Ulex europaeus agglutinin(UeA1). Binding inhibition (40–50%) was observed with10–50 mM carbohydrates and 25–50 µg ml^-1 lectins.The inhibitory effects of ConA and UeA1 is partially reversed(to 80–90% of controls) by addition of -methyl D-mannoseand L-fucose, respectively. Lectin binding to spermatial surfaceswas visualized by Fluorescein isothiocyanate (FITC) conjugatedConA, whereas carbohydrate receptors along the trichogyne andspermatium were localized with -mannosylated-FITC-albumin andL-fucosylated-FITC-albumin, respectively. These results suggestthat gamete recognition in Antithamnion sparsum is mediatedby a double-docking recognition system consisting of spermatiapossessing surface L-fucose receptors and -methyl D-mannosemoiety, and trichogynes possessing the complementary receptors. (Received December 5, 1995; Accepted April 22, 1996)     

Common mutations in the phosphofructokinase-M gene in Ashkenazi Jewish patients with glycogenesis VII--and their population frequency.

Phosphofructokinase (PFK) catalyzes the rate-limiting step of glycolysis. Deficiency of the muscle enzyme is manifested by exercise intolerance and a compensated hemolytic anemia. Case reports of this autosomal recessive disease suggest a predominance in Ashkenazi Jews in the United States. We have explored the genetic basis for this illness in nine affected families and surveyed the normal Ashkenazi population for the mutations we have found. Genomic DNA was amplified using PCR, and denaturing gradient-gel electrophoresis was used to localize exons with possible mutations. The polymorphic exons were sequenced or digested with restriction enzymes. A previously described splicing mutation, delta 5, accounted for 11 (61%) of 18 abnormal alleles in the nine families. A single base deletion leading to a frameshift mutation in exon 22 (delta C-22) was found in six of seven alleles. A third mutation, resulting in a nonconservative amino acid substitution in exon 4, accounted for the remaining allele. Thus, three mutations could account for all illness in this group, and two mutations could account for 17 of 18 alleles. In screening 250 normal Ashkenazi individuals for all three mutations, we found only one delta 5 allele. Clinical data revealed no correlation between the particular mutations and symptoms, but male patients were more symptomatic than females, and only males had frank hemolysis and hyperuricemia. Because PFK deficiency in Ashkenazi Jews is caused by a limited number of mutations, screening genomic DNA from peripheral blood for the described mutations in this population should enable rapid diagnosis without muscle biopsy.     

Production of high fructo-oligosaccharide syrup with two enzyme system of fructosyltransferase and glucose oxidase

Summary A novel two enzyme system of fructosyltransferase and glucose oxidase to enhance the content of the net fructo—oligosaccharide (FOS) fractions in the industrial production of FOS syrup from sucrose was devised. The net FOS content in the commercial FOS syrup has been limited only to 55–60 % due to the accumulation of glucose which acts as a feedback inhibitor of the fructosyltransferase. By supplementing glucose oxidase to the conventional FOS reaction system, we could convert the glucose to gluconic acid readily separable from neutral sugars by simple ion exchange operation in the next step. The simultaneous removal of glucose was proved effective in proceeding the reaction by fructosyltransferase further by relieving the product inhibition caused by glucose. By this way, we could raise the net FOS content as high as 90 %.     

A SIGNAL GLYCOPROTEIN WITH α-d-MANNOSYL RESIDUES IS INVOLVED IN THE WOUND-HEALING RESPONSE OF ANTITHAMNION SPARSUM (CERAMIALES,RHODOPHYTA)1

A variety of fluorescein isothiocyanate-labeled lectins specific for different sugar moieties were examined as probes for the wound-healing response in the filamentous red alga Antithamnion sparsum Tokida. Among them, only concanavalin A (ConA) and Lens culrinaris agglutinin (LCA), which have specificity to α-D-mannosyl residues, bound specifically to repair cells during the wound-healing process. When ConA or LCA was added at various time intervals after wounding, it first bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair cells. Intense labeling at these sites continued throughout the healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion. When added to plants prior to wounding and continually monitored, these same lectins acted as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. A crude extract solution obtained from decapitated filaments stimulated the wound-healing response, and a lectin-binding component bound strongly to a protein-binding transfer membrane. These results suggest that the labeled compound is a glycoprotein that has α-D-mannosyl residues and is similar to the repair hormone rhodomorphin found in Griffithsia pacifica Kylin.     

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.     

Interleukin-4 plus tumor necrosis factor α augments the antigenicity of melanoma cells

Immune cytokines are important regulators of the immune response to neoplastic cells. We previously reported that interleukin 4 (IL-4) and either tumor necrosis factor α (TNF) or interferon γ (IFN) synergistically inhibit melanoma cell growth and induce cell differentiation. In the present study we used various combinations of IL-4, IFN and TNF to enhance the antigenicity of melanoma cells. IL-4 plus TNF significantly increased the ability of melanoma cells to stimulate cytotoxic T cells (CTL) and act as targets of these CTL; IL-4 plus IFN was somewhat less effective, while TNF plus IFN was not as effective. IL-4 plus TNF also increased the expression of HLA class I and HLA-DR antigens on melanoma cells. The CTL lines examined in this study were CD3⁺CD4⁺ and oligoclonal. These preclinical results suggest that the immune response to melanoma whole-cell vaccines might be enhanced by pretreating vaccine cells with IL-4 plus TNF.