Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h(-1) in the CST bioreactor and between 0.111 and 0.500 h(-1) in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris-EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g(-1) dry weight (dw) as dilution rate increases from 0.027 to 0.115 h(-1). At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g(-1) dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g(-1) dw at dilution rates between 0.111 and 0.200 h(-1) to approximately 0.119 mg ATP g(-1) dw at dilution rates between 0.300 and 0.500 h(-1). This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable. (c) 1993 John Wiley & Sons, Inc.     

Developmental requirements for the ecdysoneless (ecd) locus in Drosophila melanogaster

The ecdysoneless locus in Drosophila melanogaster has been defined previously by a single conditional mutation, I(3)ecd¹, that causes an ecdysteroid deficit and larval death at the restrictive temperature, 29°C, although the primary role of the mutation in developmental processes has been unclear. Gene dosage and complementation studies reported here for ecd¹ and five nonconditional lethal alleles indicate that the ecd locus plays prezygotic and postzygotic roles essential for normal embryonic development, the successful completion of each larval molt, adult eclosion, and female fertility. The ecd locus is also required for normal macrochaete differentiation. For each observed phenotype, the severity of mutational effects was correlated with ecd mutant genotypes. In all cases, ecd¹ homozygotes were least affected. Mutants heteroallelic for ecd¹ and any one of four nonconditional recessive mutations were more severely affected than ecd¹ homozy-gotes, revealing these as hypomorphic alleles. For all phenotypic effects, mutants heteroallelic for ecd¹ and a dominant mutation (ecd^3D) were most severely affected. These individuals died during embryogenesis at 29°C and developed no macrochaetes on the dorsal thorax when transferred to 29°C during the white prepupal stage. The ecd^3D mutation also caused female semisterility in heterozygotes. Ecdysteroid regulation has been implicated previously in all the developmental processes disrupted by these ecd mutations except for macrochaete differentiation. © 1993 Wiley-Liss, Inc.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Practical applications of time-averaged restrained molecular dynamics to ligand-receptor systems: FK506 bound to the Q50R,A95H,K98I triple mutant of FKBP-13

Summary The ability of time-averaged restrained molecular dynamics (TARMD) to escape local low-energy conformations and explore conformational space is compared with conventional simulated-annealing methods. Practical suggestions are offered for performing TARMD calculations with ligand-receptor systems, and are illustrated for the complex of the immunosuppressant FK506 bound to Q50R,A95H,K98I triple mutant FKBP-13. The structure of ¹³C-labeled FK506 bound to triple-mutant FKBP-13 was determined using a set of 87 NOE distance restraints derived from HSQC-NOESY experiments. TARMD was found to be superior to conventional simulated-annealing methods, and produced structures that were conformationally similar to FK506 bound to wild-type FKBP-12. The individual and combined effects of varying the NOE restraint force constant, using an explicit model for the protein binding pocket, and starting the calculations from different ligand conformations were explored in detail.Abbreviations DG distance geometry - dmFKBP-12 double-mutant (R42K,H87V) FKBP-12 - FKBP-12 FK506-binding protein (12 kDa) - FKBP-13 FK506-binding protein (13 kDa) - HSQC heteronuclear single-quantum coherence - K_NOE force constant (penalty) for NOE-derived distance restraints - MD molecular dynamics - NOE nuclear Overhauser effect - SA simulated annealing - TARMD molecular dynamics with time-averaged restraints - tmFKBP-13 triple-mutant (Q50R,A95H,K98I) FKBP-13 - wtFKBP-12 wild-type FKBP-12     

Deoxyribonucleic acid sequence organization of a yeast plasmid.

Two-micrometer deoxyribonucleic acid (DNA), a circular plasmid of Saccharomyces cerevisiae, contains two nontandem repeated sequences which are inverted with respect to one another. These repeated sequences together account for 21% of the molecule length. Restriction endonuclease analysis and electron microscopy demonstrated the existence of two forms of 2-mum DNA differing in the orientation of the interstitial segments bounded by the inverted repeated sequences. The two forms of 2-mum DNA could result from an intramolecular reciprocal recombination between inverted repeat elements. A map containing the restriction endonuclease sites and the units of the inverted repeat has been constructed.     

Biological and biochemical studies of cells transformed by simian virus 40 temperature-sensitive gene A mutants and A mutant revertants.

The growth properties of hamster cells transformed by wild-type Simian virus 40 (SV40), by early SV40 temperature-sensitive mutants of the A complementation group, and by spontaneous revertants of these mutants were studied. All of the tsA mutant-transformed cells were temperature sensitive in their ability to form clones in soft agar and on monolayers of normal cells except for CHLA-30L1, which was not temperature sensitive in the latter property. All cells transformed by stable revertants of well-characterized tsA mutants possessed certain growth properties in common with wild-type-transformed cells at both temperatures. Virus rescued from tsA transformants including CHLA30L1 was temperature sensitive for viral DNA replication, whereas that rescued from revertant and wild-type transformants was not thermolabile in this regard. T antigen present in crude extracts of tsA-transformed cells including CHLA30L1, grown at 33 degreeC, was temperature sensitive by in vitro immunoassay, whereas that from wild-type-transformed cells was relatively stable. T antigen from revertant transformants was more stable than the tsA protein. Partially purified T antigen from revertant-transformed cells was nearly as stable as wild-type antigen in its ability to bind DNA after heating at 44 degrees C, whereas T antigen from tsA30 mutant-transformed cells was relatively thermolabile. These results further indicate that T antigen is a product of the SV40 A gene. Significantly more T antigen was found in extracts of CHLA30L1 grown to high density at the nonpermissive temperature than in any other tsA-transformed cell similarly grown. This is consistent with the suggestion that the amount of T antigen synthesized in CHLA30L1 is large enoughto allow partial expression of the transformed phenotype at the restrictive temperature. Alternatively, the increase in T antigen concentration may be secondary to one or more genetic alterations that independently affect the transformed phenotype of these cells.     

Insulin degradation by adipose tissue. Studies at several levels of cellular organization.

A systematic study of the degradation of physiological concentrations of 125I-labelled insulin was performed in intact fat-pads, isolated adipocytes and subcellular fractions of isolated adipocytes. The findings indicate that insulin is rapidly degraded to low-molecular-weight peptides and/or amino acids by the intact tissue and isolated cells. Of the total insulin-degradation products present after incubation with an intact fat-pad, 94% is recovered in the medium, indicating that these products are not retained by the cells or tissue. The plasma membranes do not degrade insulin significantly in the absence of reduced glutathione, and over 99% of the cellular degradative capacity is found in the postmicrosomal supernatant (cytosol). The cytosol degrades insulin to several labelled fragments that are intermediate in size between insulin and insulin A chain, as well as to the low-molecular-weight tissue degradation products. Inclusion of plasma membranes with cytosol accelerates the cleavage of the intermediate fragments to the size of the small products seen with the intact tissue. However, plasma membranes do not increase the initial step in the degradation of insulin when incubated with cytosol, suggesting that the insulin receptor is not involved with the direct cleavage of insulin. This study supports the hypothesis that the bulk of insulin degradation occurs in the adipocyte cytosol, where intermediate-sized fragments are generated and rapidly cleaved to smaller products by the plasma membrane and quickly released into the surrounding medium.     

Thermometry and dosimetry of heat with specific reference to the liquid-crystal optical fiber temperature probe

Summary A temperature sensing probe was designed utilizing the unique optical properties of thermotropic liquid crystals. An electronic and fiberoptic system coupled to the sensor was used to monitor temperature-dependent interactions of light (660 nm) with the liquid crystal mixture. The probe is a nonmetallic, nonperturbing thermal sensor for continuous temperature measurements of biological specimens subjected to electromagnetic fields. The advantages and disadvantages of the Liquid Crystal Optical Fiber (LCOF) temperature probe are discussed along with some typical applications where accurate dosimetry and thermometry are important parameters in experimental studies.     

Saccharomyces cerevisiae spheroplasts are sensitive to the action of diphtheria toxin.

Diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae but not the intact yeast cells. After 2 h of exposure to ca. 10(-7) M toxin, less than 1% of spheroplasts were able to regenerate into intact cells. The same high levels of toxin inhibited the rate of protein synthesis by more than 90% within 1 h, whereas RNA and DNA synthesis were not inhibited until 4 h or exposure. Both killing and protein synthesis inhibition were dependent on toxin concentration. The nature of the toxin-cell interaction was also studied by using fragments of intact toxin and mutant toxin proteins. Neither toxin fragment A nor CRM45 nor CRM197 affected spheroplasts, but CRM197 and ATP prevented the inhibitory action of intact toxin. These results suggest that toxin acts on S. cerevisiae spheroplasts in much the same manner as it acts on sensitive mammalian cells.