Hybridomas in a bioreactor cascade: modeling and determination of growth and death kinetics

Hybridomas were cultured under steady-state conditions in a series of two continuous stirred-tank reactors (CSTRs), using a serum-free medium. The substrate not completely converted in the first CSTR, was transported with the cells to the second one and very low growth rates, high death rates, and lysis of viable cells were observed in this second CSTR. These conditions are hardly accessible in a single vessel, because such experiments would be extremely time-consuming and unstable due to a low viability. In contrast to what is often observed in literature, kinetic parameters could thus be derived without the neccessity for extrapolation to lower growth rates. Good agreement with literature averages for other hybridomas was found. Furthermore, showing that the reactor series is a valuable research tool for kinetic studies under extreme conditions, the possibility to observe cell death under stable and defined steady-state conditions offers interesting opportunities to investigate apoptosis and necrosis. Additionally, a model was developed that describes hybridoma growth and monoclonal antibody production in the bioreactor cascade on the basis of glutamine metabolism. Good agreement between the model and the experiments was found.Abbreviation MAb Monoclonal antibody Nomenclature C _AConcentration of any (mol m^-3) component A D Dilution rate (s^-1) K _dDeath-rate constant (mol m^-3) K _lLysis-rate constant (mol m^-3) K _sMonod constant (mol m^-3) m Maintenance coefficient (mol cell^-1 s^-1) q Specific consumption (mol cell^-1 s^-1) or production rate t Time (s) X Cell concentration (cell m^-3) Y Yield coefficient (cell mol^-1) Greek symbols _d Specific death rate (s^-1) _l Specific lysis rate (s^-1) of viable cells _net Net specific growth (s^-1) rate _true True specific growth (s^-1) rate     

An impedimetric method for rapid screening of cosmetic preservatives

An efficient impedance method was developed for rapid evaluation of cosmetic preservatives. The method used decimal reduction time or D-value to assess preservative efficacies. The D-value, which was calculated from the plot of Log CFU ml^–1 versus time by linear regression analysis, could be obtained within 48 h. Thus, the time required for the challenge test was reduced from 4–8 weeks with the standard procedures (eg US Pharmacopeia), to 2 days with the current method. A calibration curve (r=-0.95) was established by plotting the Log CFU ml^–1 versus capacitance detection time (DT) of 108 samples. With the calibration, CFU can be estimated directly from the impedance test without plating. Two commercial biocides and several other chemicals were evaluated in a shampoo by the impedance procedure againstPseudomonas aeruginosa. The D-values obtained from the impedance test were not significantly different from those produced by the conventional plate count method. The technique was found to be particularly useful when screening a large number of compounds to find novel preservatives and synergistic preservative combinations.     

Net Uptake of Sulfate and its Transport to the Shoot in Spinach Plants Fumigated with H2S or SO2: Does Atmospheric Sulfur Affect the “Inter-Organ” Regulation of Sulfur Nutrition?*

Spinach plants (Spinacea oleracea L. cv. Estivato) were grown on nutrient solutions under deficient, normal and excess sulfate supply. In both young and mature plants net uptake of sulfate and its transport to the shoot increased with increasing sulfate supply, but both processes proceeded at a higher rate in young as compared to mature plants. The relative sulfate transport, i.e. the relative amount of the sulfate taken up that is transported to the shoot, decreased with increasing sulfate supply. Apparently, net uptake of sulfate is not strictly controlled by the sulfur demand of the shoot, but xylem loading appears to counteract excess transport of sulfate to the shoot. Fumigation with H₂S or SO₂ reduced net uptake of sulfate by the roots in sulfur-deficient plants and absolute as well as relative sulfate transport to the shoot independent of the three sulfate levels supplied to the plant. At the same time thiol contents of the shoot and the root were enhanced by fumigation with H₂S and SO₂. These findings are consistent with the idea that thiols produced in the leaves can mediate demand-driven control of sulfate uptake by the roots and its transport to the shoot.     

Influence of Early Season Moxidectin Treatments on Acquisition of Immunity to Ostertagia ostertagi in Calves

Moxidectin, a newly developed endectocide, has previously been demonstrated to have a high efficacy against gastrointestinal nematodes in cattle (Ranjan et al. 1992, Scholl et al. 1992). In a field trial (Yang, Satrija and Nansen, unpublished data) matched groups of 8 first-season Black Pied Friesian heifer calves, 6-8 months of age, were turned out in early May 1992 on a permanent pasture contaminated with trichostrongyles. The groups were grazed separately on comparable plots until they were housed in mid October 1992. Group Β was treated with moxidectin at turnout, and group C was treated at turnout and again 8 weeks after turnout. A third group served as non-treated controls (group A). The results showed that the 2 early season prophylactic treatments (group C) reduced the late season herbage trichostrongyle larval infectivity with 80 to 90%, whereas the reduction accomplished by only 1 treatment at turnout (group B) was much less. Group C excreted significantly lower numbers of strongyle eggs than groups A and B. The non-treated controls showed clinical signs of trichostrongylosis in late September, which coincided with a steep rise in serum pepsinogen levels reaching an average peak level of 3.5 IU tyrosine litre-1. A few animals in group Β showed softening of faeces particularly in the very late season. In contrast, animals receiving 2 moxidectin treatments remained in a clinically good condition throughout the season.     

Inhibition of rabbit sperm acrosomal enzymes by gossypol

The effect of gossypol on the activities of 10 acrosomal enzymes of the rabbit sperm was evaluated. Acrosin, Azocoll proteinase, neuraminidase, and arylsulfatase were significantly inhibited or completely inactivated by 12–76 μM gossypol. Hyaluronidase, β-glucuronidase, and acid phosphatase were inhibited only at a higher concentration of gossypol (380 μM). Phospholipase C, alkaline phosphatase, and β-N-Acetyl glucosaminidase were not inhibited even at 380 μM gossypol. Gossypol was found to be a noncompetitive inhibitor of arylsulfatase with a Ki of 120 μM. The inhibition was reversible and dose-dependent. As the acrosomal enzymes were more sensitive to the inhibition by gossypol compared to sperm enzymes involved in glycolysis or energy production, these assays may serve as a more reliable indicator for monitoring the occurence of gossypol-induced sterility. © 1995 Wiley-Liss, Inc.     

Preliminary evidence of endotoxic activity of Bilophila wadsworthia

In this study we describe two properties of the Gram-negative bacterium Bilophila wadsworthia, namely the ability to clot Limulus lysate and the capacity to induce the production of tissue factor-like procoagulant activity by human mononuclear cells in vitro. Although exhibited at a lower degree when compared with those of typical Gram-negative bacteria or Gram-negative endotoxin those activities may account in part for Bilophila's pathogenicity. The capacity indeed to induce fibrin formation through the interaction with mononuclear cells suggests one mechanism by which the microorganism might cause abscess formation in the host. Moreover, since this activity is dependent on the number of Bilophila interacting with mononuclear cells, we hypothesize that this biological activity is closely influenced by growth environment.