Intracellular flux analysis applied to the effect of dissolved oxygen on hybridomas

Quantitative estimates of intracellular fluxes and measurements of intracellular concentrations were used to evaluate the effect of dissolved oxygen (DO) concentration on CRL 1606 hybridoma cells in batch culture. The estimates of intracellular fluxes were generated by combining material balances with measurements of extracellular metabolite rates of change. Experiments were performed at DO levels of 60% and 1% air saturation, as well as under oxygen-limited conditions. Cell extracts were analyzed to evaluate the effect of DO on the intracellular concentrations of the glutamate dehydrogenase reactants, as well as the redox state of the pyridine nucleotides in the cytosol and mitochondria. The relationship between cell density and pyridine nucleotide redox state was also investigated. Dissolved oxygen concentration had a significant effect on nitrogen metabolism and the flux through glutamate dehydrogenase was found to reverse at low DO, favoring glutamate formation. The NAD in the cytosol and mitochondria was more reduced under low DO conditions while the cytosolic NAD was more oxidized at low DO. Cytosolic NAD was reduced at higher cell densities while the redox states of cytosolic NADP and mitochondrial NAD did not exhibit significant variation with cell density. These results point to the fundamental role of the intracellular oxidation/reduction state in cell physiology and the possibility of controlling physiological processes through modulation of the dissolved oxygen level or the oxidation/reduction potential of the culture.     

Malaria-induced reduction of fecundity during the first gonotrophic cycle of Anopheles Stephensi mosquitoes

Abstract. Anopheles stephensi mosquitoes which had fed upon mice infected with Plasmodium yoelii nigeriensis malaria parasites produced significantly fewer eggs than mosquitoes fed on an uninfected mouse. Fecundity reduction was more pronounced when the bloodmeal contained malaria gametocytes and the mosquitoes developed oocysts. Egg production and haematin excretion were correlated for uninfected bloodfed mosquitoes; the presence of P.y. nigeriensis in the blood affected this relationship. Reduced fecundity was associated with a significant reduction of bloodmeal size (measured by haematin excretion) in mosquitoes which ingested gametocytaemic blood. The bloodmeal size in mosquitoes fed on parasitaemic blood without gametocytes was not significantly reduced. The use of haematin assays for determination of bloodmeal size in mosquitoes is discussed.     

Scheduling algorithms for a two-machine flexible manufacturing system

The flexible manufacturing system (FMS) considered in this paper is composed of two CNC machines working in series—a punching machine and a bending machine connected through rollers acting as a buffer system of finite capacity. The main difference between the present problem and the standard two-machine flow shop problem with finite intermediate capacity is precisely the buffer system, which in our problem consists of two stacks of parts supported by rollers: the first stack contains the output of the punching machine, while the second stack contains the input for the bending machine. When the second stack is empty, the first stack may be moved over. Furthermore, the capacity of each stack depends on the particular part type being processed. The FMS can manufacture a wide range of parts of different types. Processing times on the two machines are usually different so that an unbalance results in their total workload. Furthermore, whenever there is a change of the part type in production, the machines must be properly reset—that is, some tools need to be changed or repositioned. A second important difference between the present problem and the usual two-machine flow shop problem is the objective. Given a list ofp part types to be produced in known quantities, the problem considered here is how to sequence or alternate the production of the required part types so as to achieve various hierarchical targets: minimize the makespan (the total time needed to complete production) and, for instance, compress the idle periods of the machine with less workload into a few long enough intervals that could be utilized for maintenance or other reasons. Although Johnson's rule is optimal in some particular cases, the problem addressed in the paper isNP-hard in general: heuristic procedures are therefore provided.     

Protective effects of anti-antiidiotypic IgE antibodies obtained from an IgE monoclonal antibody specific for a 26-kilodalton Schistosoma mansoni antigen

A rat IgE mAb specific for larval Ag (26 kDa, 56 kDa) has been shown to protect rats against Schistosoma mansoni infection. Immunizations of Lou/M rats performed with this IgE (Ab1) induced the production of antiidiotypic antibodies (Ab2). Moreover, after this Ab2 production, anti-antiidiotypic antibodies (Ab3) were revealed. The screening of Ab3 isotypes showed the presence of IgG Ab3 and more interestingly of IgE Ab3, i.e., the same isotype as Ab1. These IgE and IgG antibodies recognized predominantly the 26-kDa Ag and were cytotoxic for schistosomula in the presence of platelets for IgE Ab3 and eosinophils for IgG Ab3. Both IgE and IgG Ab3 conferred by passive transfer protective immunity to infected rats (up to 50%). Thus the immunization with an IgE mAb led in part to the production of Ab3 of the same isotype as Ab1. In conclusion, these results suggest that the isotype selection of the antibodies of the third generation (Ab3) might be influenced by the Ab1. The respective role of the idiotope and isotype of Ab1 in isotype regulation is discussed.     

Effect of medium composition, agitation and the presence of EDTA on the antimicrobial activity of cryptolepine

The antimicrobial activity of cryptolepine is influenced by the type of medium employed, agitation and the presence of non-inhibitory concentrations of EDTA. The use of Mueller–Hinton broth (MHB), iso-sensitest broth and tryptone soya broth (TSB) produced lower minimum inhibitory concentrations (MICs) for some of the test organisms compared with nutrient broth or yeast dextrose broth (YDB). For example, a fourfold drop in MIC was recorded for Saccharomyces cerevisiae in MHB compared with the same organism tested in YDB. Agitation of the broths during incubation nearly always produced lower MICs for the bacteria, an eightfold decrease in MIC being recorded for Escherichia coli cultured in nutrient broth with agitation compared with a statically maintained culture. A non-inhibitory concentration (10⁻³ mol l⁻¹) of disodium EDTA enhanced the antimicrobial activity of cryptolepine. Against E. coli NCTC 11560, an eightfold decrease in MIC and minimum bactericidal concentration (MBC) was recorded when tested in the presence of EDTA.     

Association of LHIα(B870) Polypeptide with Phospholipids During Insertion in the Photosynthetic Membrane of an LHII− Mutant of Rhodobacter capsulatus

Membranes from in vivo labeled cells of Rhodobacter capsulatus U43[pTX35] grown photosynthetically carried 60% of the [³²P]-Pi in the “heavy” fraction (HM) after sucrose gradient sedimentation. Metal-chelating chromatography of either “heavy” or “light” (LM) membrane fractions rendered similar Bchl-protein complex profiles after octyl-glucoside treatment, including most of the radioactivity in the same corresponding elution fraction (F II). Similar labeling distribution of pigment-protein complexes was obtained for membranes of dark-grown cells induced by lowering oxygen tension. Fractions derived from HM showed highly labeled LHIα, whereas the same complex from LM was essentially [³²P]-Pi-free, as revealed by SDS-PAGE followed by autoradiography. Phospholipid analysis showed a similar pattern for membranes isolated from cells photosynthetically or semiaerobically grown, being the most abundant: phosphatidylglycerol, phosphatidylethanolamine, cardiolipin, and phosphatidylcholine. Part of the phospholipids from HM comigrated with LHIα during SDS-PAGE and dissociated from the complexes only after solvent extraction and hydrophobic chromatography. However, a small amount remained always attached to LHIα, indicating an unusual strong interaction. These results suggest the existence of two operationally defined membrane regions carrying LHIα complexes differing in phosphorylation status and protein-phospholipid interaction. Received: 10 August 1996 / Accepted: 10 September 1996