IgG production kinetics in serum free media

Summary A murine hybridoma (455) was cultured in four different serum free media formulations, and a newborn calf serum supplemented medium was used as a basis of comparison. The serum supplemented medium supports a higher cell growth rate and results in a higher IgG titer. However, the antibody secretion rate on a per cell basis is higher in the serum free media, indicating that serum could be inhibitory to antibody secretion. The results identify the possibility of a least eliminating serum during the monoclonal antibody production phase.     

Photoaffinity labeling of platelet activating factor binding sites in rabbit platelet membranes

A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn- glycero-3-phosphocholine ([125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC50 value of 3.2 +/- 1.9 nM as compared to 0.40 +/- 0.25 nM for PAF. Specific binding of [125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (Kd) of 2.4 +/- 0.7 nM and a receptor density (Bmax) of 1.1 +/- 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr = 52,000.     

Tegumental glucose permeability in male and female Schistosoma mansoni

Tegumental hexose transporters have been kinetically characterized in mated and separated male and female Schistosoma mansoni 8-12 wk postinfection. Significant gender-specific differences in Km and Vmax were observed. In mated males, the estimated constants (mean +/- SE) were: Km = 0.63 +/- 0.31 mM, Vmax = 0.93 +/- 0.44 nmol/mg worm water/min, and the Kd = 0.25 +/- 0.09 microliter/mg worm water/min. In mated females the kinetics were: Km = 0.99 +/- 0.40 mM, Vmax = 1.22 +/- 0.42 nmol/mg worm water/min, and Kd = 0.60 +/- 0.14 microliter/mg worm water/min. The influx of 2-deoxy-D-glucose and 3-O-methylglucose has been similarly characterized; these analogs share the same glucose transporter in male and female schistosomes. 2-Deoxy-D-glucose has a higher affinity, and 3-O-methylglucose a lower affinity, than does glucose. Because mated male schistosomes supply glucose to female partners, similarities between the free glucose concentration of the male and the affinity of the transporter determined for mated female schistosomes suggest that male-to-female transfer may be a potentially rate-limiting step in glucose utilization by the female. Permeability x surface are (PS) products and Vmax/Km ratios were significantly elevated in mated schistosomes, suggesting that the transporter is primarily localized to the dorsal surface of the male. Gender- and mating-specific analyses of PS products indicate that tegumental permeability to glucose is significantly increased in mated schistosomes, and compares very favorably to that of the host liver.     

Operation and pressure distribution of immobilized cell hollow fiber bioreactors

It has been cited in the literature on hollow fiber systems that pressure gradients persist, and the transmembrane flux of the hollow fiber system is dependent on the pattern of the pressure gradients. The pattern can be used to its advantage in immobilized enzyme systems. However, with immobilized living cell systems, the pressure gradients lead to a nonuniform environment within the hollow fiber cartridge and not necessarily favorable results. This article provides pertinent pressure-drop data on hollow fiber cartridges which are in flow configurations typical of immobilized cell culture work. The results illuminate operational problems that may arise in the culture of either anchorage dependent or independent cells. Possible solutions with crossflow systems are suggested.     

Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.     

Endothelin-Mediated Calcium Response and Inositol 1,4,5-Trisphosphate Release in Neuroblastoma-Glioma Hybrid Cells (NG108-15): Cross Talk with ATP and Bradykinin

Abstract: Addition of endothelins (ETs) to neuroblastomaglioma hybrid cells (NG108-15) induced increases in cytosolic free Ca²⁺ ([Ca²⁺]_i) levels of labeled inositol monophosphates and inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]. The increases in [^Ca2+]_i elicited by the three ETs (ET-1, ET-2, and ET-3) were transient and did not show a sustained phase. Chelating extracellular Ca²⁺ in the medium by adding excess EGTA decreased the ET-mediated Ca²⁺ response by 40-50%. This result indicates that a substantial portion of the increase in [Ca²⁺]_i was due to influx from an extracellular source. However, the increase in [Ca²⁺]_i was not affected by verapamil or nifedipine (10^?5M). A rank order potency of ET-1 ET-2 ET-3 is shown for the stimulated increase in [Ca²⁺]_i, as well as labeled inositol phosphates, in these cells. ATP (10^?4M) and bradykinin (10^?7M) also induced the increases in [Ca²⁺]_i and Ins(1,4,5)P₃ in NG108-15 cells, albeit to a different extent. When compared at 10^?7M, bradykinin elicited a five- to sixfold higher increase in the level of Ins(1,4,5)P₃, but less than a twofold higher increase in [Ca²⁺]_i than those induced by ET-1. Additive increases in both Ins(1,4,5)P₃ and [Ca²⁺]_i were observed when ET-1, ATP, and bradykinin were added to the cells in different combinations, suggesting that each receptor agonist is responsible for the hydrolysis of a pool of polyphosphoinositide within the membrane. ET-1 exhibited homologous desensitization of the Ca²⁺ response, but partial heterologous desensitization to the Ca²⁺ response elicited by ATP. On the contrary, ET-1 did not desensitize the response elicited by bradykinin, although bradykinin exhibited complete heterologous desensitization to the response elicited by ET-1. Taken together, these results illustrate that, in NG108-15 cells, a considerable amount of receptor cross talk occurs between ET and other receptors that transmit signals through the polyphosphoinositide pathway.     

Engineered nickel bioaccumulation in Escherichia coli by NikABCDE transporter and metallothionein overexpression

Mine wastewater often contains dissolved metals at concentrations too low to be economically extracted by existing technologies, yet too high for environmental discharge. The most common treatment is chemical precipitation of the dissolved metals using limestone and subsequent disposal of the sludge in tailing impoundments. While it is a cost-effective solution to meet regulatory standards, it represents a lost opportunity. In this study, we engineered Escherichia coli to overexpress its native NikABCDE transporter and a heterologous metallothionein to capture nickel at concentrations in local effluent streams. We found the engineered strain had a 7-fold improvement in the bioaccumulation performance for nickel compared to controls, but also observed a drastic decrease in cell viability due to metabolic burden or inducer (IPTG) toxicity. Growth kinetic analysis revealed the IPTG concentrations used based on past studies lead to growth inhibition, thus delineating future avenues for optimization of the engineered strain and its growth conditions to perform in more complex environments.