Factors governing lactic acid formation in long term cultivation of hybridoma cells

Summary Effect of glucose concentration and pH on lactic acid formation was investigated in batch cultures of a hybridoma cell line. Lactate formation increases with growth rate. High glucose concentration leads to extensive lactate formation only during growth phase and not during stationary phase. Lactate formation also may serve to regulate extracellular pH to pH 6.8, provided conditions are favorable to maintain viability and if sufficient nutrients are present in the medium.     

Factors influencing the observed half-lives of specific synthetic capacities in Saccharomyces cerevisiae.

We have identified a variety of factors affecting the stability of allophanate hydrolase-specific and gross cellular protein synthetic capacities. These synthetic capacities have been extrapolated by many laboratories to represent functional messenger RNAs. Synthetic capacity turnover rates that we measured were greater in diploid organisms than in haploid strains and were proportional to the temperature of the culture medium. The stability of allophanate hydrolase-specific synthetic capacity was not influenced by alterations in the nitrogen source provided in the culture medium, but was increased up to 15-fold by the total inhibition of protein synthesis. Cultures in which protein synthesis was inhibited as little as 20% exhibited hydrolase-specific synthetic capacities more than 2-fold greater than those observed in the absence of inhibition.     

Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides

Site-specific conjugation of small molecules and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogeneous materials that could have improved biological properties. We describe for the first time chemical reduction and oxidation methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purification with hydrophobic interaction chromatography followed by analysis using reversed-phase HPLC and capillary electrophoresis. These analytical methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential reduction of heavy-light chain disulfides, while reoxidation of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60-90%, allowing for control of the distribution of molecular species. The resulting conjugates are highly active both in vitro and in vivo and are well tolerated at efficacious doses.     

Factors governing the epidemiology of Histoplasma capsulatum in soil

The growth ofH. capsulatum in soil is markedly affected by soil pH above 10 and below 5. No growth was observed on soil cultures outside these values. There was no definite pH range within these values in which the fungus grew more abundantly. A chemical analysis of natural soils from whichH. capsulatum had been isolated failed to show any common chemical factors that would explain the presence of the fungus in the soils.

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Zusammenfassung Das Wachstum desH. capsulatum im Erdboden ist durch das pH des Bodens über 10 und unter 5 wesentlich beeinflußt. Kein Wachstum ist in Bodenkulturen außerhalb dieser Werte beobachtet worden. Es fand sich keine bestimmte Grenze innerhalb dieser Werte, in welcher der Pilz ein üppigeres Wachstum gezeigt hätte. Eine chemische Analyse des naturlichen Bodens, von welchemH. capsulatum isoliert ist, hat keine gemeinsamen Faktoren gezeigt, der die Gegenwart des Pilzes im Boden hätte erklären konnen.

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Paper read at the Eighth International Congresses for Tropical Medicine and Malaria, September 1968, Teheran (Iran).     

Hairpin formation in synthetic oligonucleotides

The structure and dynamics of the homologous series of the (partly) self-complementary DNA fragments, d(ATCCTATnTAGGAT) n = 0-7, were investigated in a combined NMR, T-jump, and optical melting study. It is shown that all compounds in the series may adopt hairpin like conformations, even for n less than 3, although for these smaller n values this only occurs in significant amounts at relatively low concentrations (approximately 10 microM). The enthalpy change accompanying the hairpin-coil melting transition turns out to depend on the number of intervening thymidines, n. It is shown that this does not mean that the enthalpy of loop closure is significantly different from zero, but that loop formation stabilizes the base pair closing the loop. The results indicate that for DNA the optimal loop consists of four or five residues. The observation that hairpins are formed for n less than 3 and that the stability of DNA hairpins is at its maximum for loop lengths of four to five residues is at variance with earlier findings for RNA. In the latter case the optimal loop size consists of six to seven residues, whereas for less than three intervening residues only, dimer, and no hairpin formation, was observed [17, 20]. A direct comparison with RNA behaviour was made by studying r(AUCCUAUT4UAGGAU), T = ribothymidine. In contrast to its DNA analogue, d(ATCCTAT4TAGGAT), the ribo-fragment forms a dimer as well as a hairpin at low (10 microM) concentrations. With the thermodynamic melting parameters deduced from the present experiments the differences between DNA and RNA melting behaviour can be explained.     

Factors governing the assembly of cationic phospholipid-DNA complexes

The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction between large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic strength, although the heat absorbed was slightly larger at the higher ionic strength. The energetic driving force for DNA-EDOPC association is thus an increase in entropy, presumably due to release of counterions and water. The estimated minimum entropy gain per released counterion was 1.4 cal/mole- degrees K (about 0.7 kT), consistent with previous theoretical predictions. All experimental approaches revealed significant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa. When EDOPC vesicles were titrated with DNA at physiological ionic strength, particle size increased, vesicles ruptured, and membrane lipids became mixed as the amount of DNA was added up to a 1.6:1 (+:-) charge ratio. This charge ratio also corresponded to the calorimetric end point. In contrast, when lipid was added to DNA, vesicles remained separate and intact until a charge ratio of 1:1 (+:-) was exceeded. Under such conditions, the calorimetric end point was 3:1 (+:-). Thus it is clear that fundamental differences in DNA-cationic lipid complexes exist, depending upon their mode of formation. A model is proposed to explain the major differences between these two situations. Significant effects of ionic strength were observed; these are rationalized in terms of the model. The implications of the analysis are that considerable control can be exerted over the structure of the complex by exploiting vectorial preparation methods and manipulating ionic strength.     

NMR solution structure of alpha-conotoxin ImI and comparison to other conotoxins specific for neuronal nicotinic acetylcholine receptors

Alpha-Conotoxins, peptides produced by predatory species of Conus marine snails, are potent antagonists of nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels involved in synaptic transmission. We determined the NMR solution structure of the smallest known alpha-conotoxin, ImI, a 12 amino acid peptide that binds specifically to neuronal alpha7-containing nAChRs in mammals. Calculation of the structure was based on a total of 80 upper distance constraints and 31 dihedral angle constraints resulting in 20 representative conformers with an average pairwise rmsd of 0.44 A from the mean structure for the backbone atoms N, Calpha, and C' of residues 2-11. The structure of ImI is characterized by two compact loops, defined by two disulfide bridges, which form distinct subdomains separated by a deep cleft. Two short 310-helical regions in the first loop are followed by a C-terminal beta-turn in the second. The two disulfide bridges and Ala 9 form a rigid hydrophobic core, orienting the other amino acid side chains toward the surface. Comparison of the three-dimensional structure of ImI to those of the larger, 16 amino acid alpha-conotoxins PnIA, PnIB, MII, and EpI-also specific for neuronal nAChRs-reveals remarkable similarity in local backbone conformations and relative solvent-accessible surface areas. The core scaffold is conserved in all five conotoxins, whereas the residues in solvent-exposed positions are highly variable. The second helical region, and the specific amino acids that the helix exposes to solvent, may be particularly important for binding and selectivity. This comparative analysis provides a three-dimensional structural basis for interpretation of mutagenesis data and structure-activity relationships for ImI as well other neuronal alpha-conotoxins.     

Factors governing macrozoobenthic assemblages in perennial springs in north-western Switzerland

Springs are important freshwater habitats that provide refuge for many rare species. In this study, the fauna and abiotic parameters of 20 perennial springs in north-western Switzerland were investigated. Correlation of abiotic and macrozoobenthos data showed that physicochemical parameters had little impact on macrozoobenthic composition, whereas specific substrate parameters strongly influenced the composition of the macrofauna. Surprisingly, nonmetric multidimensional scaling did not reveal a grouping of springs with similar substrate composition or macrozoobenthic assemblages. However, discharge was identified as the factor significantly determining substrate and the composition of macroinvertebrate assemblages. This justifies the hypothesis that, variation in discharge is the disturbance factor governing the macrofaunal composition temporally and spatially within and between patches. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Spatial regulation of histidine kinases governing biofilm formation in Bacillus subtilis

Bacillus subtilis is able to form architecturally complex biofilms on solid medium due to the production of an extracellular matrix. A master regulator that controls the expression of the genes involved in matrix synthesis is Spo0A, which is activated by phosphorylation via a phosphorelay involving multiple histidine kinases. Here we report that four kinases, KinA, KinB, KinC, and KinD, help govern biofilm formation but that their contributions are partially masked by redundancy. We show that the kinases fall into two categories and that the members of each pair (one pair comprising KinA and KinB and the other comprising KinC and KinD) are partially redundant with each other. We also show that the kinases are spatially regulated: KinA and KinB are active principally in the older, inner regions of the colony, and KinC and KinD function chiefly in the younger, outer regions. These conclusions are based on the morphology of kinase mutants, real-time measurements of gene expression using luciferase as a reporter, and confocal microscopy using a fluorescent protein as a reporter. Our findings suggest that multiple signals from the older and younger regions of the colony are integrated by the kinases to determine the overall architecture of the biofilm community.     

Factors governing the evolution of eusociality through kin selection

The evolution of eusociality through kin selection was analyzed by simple population genetical models. Models were solved analytically with no approximation. The main results are

1. Sex ratio in reproductives in a colony of haplodiploid species does not affect the direction of evolution, contrary to the hypothesis ofTrivers andHare (1976). Female biased sex ratio increases the rate of evolution irrespective of its direction.
2. The only factor that determines the direction of evolution is the balance of benefit and cost of altruism of workers.
3. The value of ratio of benefit to cost of altruism of workers when the change of gene frequency of altruistic allele does not take place is unity in both haplodiploid and diploid species. There is no theoretical reason that the eusociality through kin selection evolves more easily in haplodiploidy than in diploidy, contrary to the hypotheses ofHamilton (1964) andTrivers andHare (1976).
4. The larger the colony size is, the lower the rate of evolution is irrespective of its direction.

It was concluded that discussion on the evolution of altruism which depended on only the values of the degrees of relatedness is misleading. The importance of life history structure, oviposition of workers and number of relating gene(s) in the evolution of eusociality were discussed.     

Factors governing the production of juvenile Atlantic salmon in Scottish streams

Data are presented to show how the number and growth of juvenile salmon in streams in the Scottish Highlands are influenced by various physical (temperature, water chemistry, depth and velocity, type of substratum) and biotic (food resources, competition, recruitment) factors.     

Factors governing nutrient status of mountain lakes in the Tatra Mountains

1. Nutrient and chlorophyll a levels, and bacterial numbers of 84 glacial lakes in the Tatra Mountains (Slovakia and Poland, Central Europe) were determined to assess the impact of catchment vegetation and water acidity on lake trophic status. 2. Catchment vegetation was the crucial factor governing nutrient content of lakes. 3. Concentrations of organic carbon, organic nitrogen, and chlorophyll a, and bacterial numbers were tightly correlated with total phosphorus (TP) content. Their levels were the highest in forest lakes, then decreased in alpine lakes with decreasing amount of catchment vegetation and soil cover, and were the lowest in lakes situated in bare rocks. 4. The above pattern was further modified by lake water acidity. Concentrations of TP, organic carbon, and chlorophyll a were lower in alpine lakes with pH between 5 and 6 than in more or less acid alpine lakes. Zooplankton was absent in all alpine lakes with pH between 5 and 6. 5. Nitrate concentrations followed an inverse trend to TP; lowest values were in forest lakes, then increased with decreasing amount of catchment soils and vegetation. Within the lakes of the same type of catchment vegetation, nitrate concentrations were negatively correlated to TP. N‐saturation of catchment areas and lake primary production were dominant processes controlling nitrate levels in lakes and nitrate contribution to lake acidification.