Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.     

Improving cultivated oats (A vena sativa L.) with alleles for vegetative growth index from A. sterilis L.

Summary Ten Avena sterilis L. lines of Mediterranean origin were crossed with six A. sativa L. cultivars from the North Central USA. Additionally, six intervarietal crosses were made among the A. sativa cultivars. F₂- derived lines from each cross type (interspecific and intraspecific) were evaluated for transgressive segregation for grain yield and several vigor traits. Mean percentages of transgressive segregates one LSD_0.05 above the high parent for vegetative growth index and biomass were 9.0% and 9.8%, respectively, from interspecific crosses, but only 4.5% and 2.9%, respectively, from intraspecific crosses. However, there were two and a half times more high transgressive segregates for grain yield from intra than from interspecific crosses. The maximum vegetative growth index among segregates from interspecific crosses was 0.2 q/day/ha greater than the highest segregate from intraspecific crosses. However, mean harvest index was reduced materially by the introgression of A. sterilis germplasm. Because there was no genetic association between vegetative growth index and harvest index, however, it should be possible to improve both harvest index and vegetative growth index and, thus, the grain yield of cultivated oats.Journal Paper No. J-11228 of the Iowa Agric. and Home Econ. Exp. Stn., Ames, IA 50011. Project 2447     

Genetic variation for grain yield and related traits in sorghum introgression populations

Summary Each of two sorghum (Sorghum bicolor (L.) Moench) cultivars were crossed with representatives of three wild sorghum races. Backcross-derived sorghum populations containing 3.125 to 50% wild germplasm were evaluated for grain yield, 100-kernel weight, days to flower, and plant height. Population means increased linearly with backcrossing for kernel weight, increased curvilinearly for grain yield, decreased curvilinearly for plant height, and changed erratically for days to flower. For all traits, the relationship between genetic variance and level of backcrossing deviated significantly from that expected based on an additive model. Genetic variance usually reached a maximum in the BC₁ or BC₂. The BC₁ genetic variance for grain yield, averaged over matings, was twice as large as the average BC₀ genetic variance. An epistatic model involving gene regulation is proposed as a plausible explanation for the results.Joint contribution of USDA-ARS, and Journal Paper No. J-11101 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2447     

Synthesis of undecagold cluster molecules as biochemical labeling reagents. 1. Monoacyl and mono[N-(succinimidooxy)succinyl] undecagold clusters

This paper describes the synthesis and characterization of succinyl, phthalyl, and N-(succinimidooxy)-succinyl derivatives of the undecagold cluster complex tricyanoheptakis[4,4',4"-phosphinidynetris(benzenemethana mine)]undecagold, 1, molecular formula Au11(CN)3[P(C6-H4CH2NH2)3]7. These are useful as electron-dense reagents for labeling biological structures in preparation for electron microscopic analysis. Limited reaction of 1 with succinic or phthalic anhydrides produces a mixture of mono-, bis-, etc. (N-succinyl)-1 or (N-phthalyl)-1, which can be separated by anion-exchange chromatography at pH 11.5. Yields of monoacylated derivatives can be maximized by controlling the ratio of succinic or phthalic anhydride to 1. The remaining 20 primary amino groups can be dialkylated or acetylated, blocking their participation in further chemical modifications of the carboxylic functional group introduced in the succinylation or phthalylation of 1. These carboxyl groups can be activated as N-hydroxysuccinimido esters, which are acylating derivatives of 1. An example is mono[N-(succinimidooxy)-succinyl]icosa(N,N-dimethyl)-1 whose synthesis is described. Bis- and tris(N-succinyl) and -(N-phthalyl) derivatives of 1 are also produced and isolated in usable quantities.     

Association of the CAMP phenomenon in Actinobacillus pleuropneumoniae with the RTX toxins ApxI, ApxII and ApxIII

Abstract A non-hemolytic mutant of Actinobacillus pleuropneumoniae serotype 5 has a deletion spanning the entire apxI operon. Therefore it does not produce ApxI and is unable to secrete ApxII. This mutant also has lost the co-hemolytic CAMP effect which is characteristic of the species A. pleuropneumoniae . The CAMP effect is restored when the mutant is complemented in trans by the apxIBD genes cloned in a broad host range vector, thus permitting secretion of ApxII, or when the entire apxI operon is cloned in the mutant, thus restoring the original toxin phenotype ApxI⁺ ApxII⁺. When the toxins ApxI, ApxII or ApxIII individually are expressed and secreted from E. coli harboring recombinant plasmids containing the genes apxICA and apxIBD or apxIICA and apxIBD or apxIIICABD , respectively, the distinct CAMP phenomenon is produced by the recombinant strains. The CAMP phenomenon is strongest by the recombinant E. coli strain expressing the non-hemolytic ApxIII, somewhat less when ApxI is expressed, and weak when ApxII is expressed. In A. pleuropneumoniae the CAMP phenomenon is also strongest in those serotypes which express ApxIII. The CAMP phenomenon of A. pleuropneumoniae is assumed to be directly caused by any of the RTX-toxins ApxI, ApxII or ApxIII. A previously reported gene from A. pleuropneumoniae , named cfp or hlyX , which provides E. coli strains with a hemolytic character and a CAMP phenomenon, shows high similarity to the E. coli global regulation gene fnr , and which is able to complement a Δfnr mutant. This gene is assumed to have a regulatory effect on the expression of yet unknown genes giving the recombinant E. coli strains a hemolytic and CAMP phenomenon like appearance.     

Der Zusammenhang zwischen Lokomotionsweise,Begattungsstellung und Penislänge bei Säugetieren1

Penis length, copulation and locomotion: Their relationship to each other in Mammals A relationship between the mode of locomotion, copulatory position and length of the male copulative organ has been found in all groups of Mammalia. In this paper particular emphasis is given to the orders of the Testiconda, while the Testiphaena receive only brief account (for explanation of terms see Frey 1991 a). Results from my previous antomical study are utilized that are essential for assessing the respective modes of locomotion (Frey 1991b). Information on penis length and copulatory position are taken from the literature and evaluated. Small and middle-sized Testiphaena, which represent the majority of mammals, are capable of a dynamic sagittal bending in the trunk, which is evidenced both in the galloping mode of locomotion and in the usual mammalian copulatory position (mounting). A sagittal bending of the trunk enables the male to bring his genital region into close proximity of the female's genital opening. In this case, a short penis is sufficient to ensure sperm transfer. The construction of the trunk in the Testiconda (excepting the Hyracoidea) entirely or nearly entirely prevents the sagittal bending. This is largely due to rigidity of the lumbar region or insufficient (dynamic) muscular control. The immobilization and/or the functional weakness of the lumbar region are derived from different anatomical conditions. 1. Shortening of the lumbar region, e.g., Tachyglossidae, Elephantidae, Sirenia; 2. Tightening of the lumbar region by tendons and muscles, e. g., Macroscelididae, Cetacea; 3. Xenarthry of the lumbar vertebrae and a double connection of the pelvis on the vertebral column, e. e., Bradypodidae, Myrmeco-phagidae; 4. The lumbar region may be flexible but the hypaxial muscles too short and weak, e. g., Tenrecinae, Soricidae, Erinaceidae. Lumbar rigidity or insufficient muscular control in the lumbar region, resulting from any of these conditions, makes copulation difficult by restricting close approximation of the male and female genitals. Most Testiconda compensate for this by having a long penis. The same also applies for large-sized Testiphaena. Although these animals have retained the ability to gallop, the flexibility of their trunk is restricted and mostly localized in a single joint due to the great body mass and the constructive constraints necessary for increased stability. A long penis is not the only way to compensate for the absence of sagittal flexion. A phylogenetic change in the copulatory posture also solves the problem as seen in the Myr-mecophagidae and Bradypodidae (both Testiconda) which are equipped with a secondarily short-enedpenis. The permanently aquatic testicondid mammals (Sirenia and Cetacea) cannot copulate in the usual mammalian mount position. This is largely due to the phylogenetic reduction and reconstruction of the extremities as well as the secondary evolution of a powerful tail. Sagittal movements of the heavily musculated tail act upon the more or less rigid trunk to provide for a “rear drive” locomotion. Both a change in the copulatory position and a long penis were necessary for these aquatic mammals. The only Testiconda in which a marked dynamic sagittal flexibility of the trunk is developed -the Hyracoidea - are characterized by a short penis. On the whole, the relationship between the mode of locomotion, copulatory position and penis length within the mammalia is confirmed. A rigid lumbar region as opposed to a sagittally flexible, presumably represents the primitive condition among mammals. All Testiconda have     

Electron crystallographic analysis of two-dimensional streptavidin crystals coordinated to metal-chelated lipid monolayers.

Coordination of individual histidine residues located on a protein surface to metal-chelated lipid monolayers is a potentially general method for crystallizing proteins in two dimensions. It was shown recently by Brewster angle microscopy (BAM) that the model protein streptavidin binds via its surface histidines to Cu-DOIDA lipid monolayers, and aggregates into regularly shaped domains that have the appearance of crystals. We have used electron microscopy to confirm that the domains are indeed crystalline with lattice parameters similar to those of the same protein crystallized beneath biotinylated lipid monolayers. Although BAM demonstrates that the two-dimensional protein crystals grown via metal chelation are distinct from the biotin-bound crystals in both microscopic shape and thermodynamic behavior, the two crystal types show similar density projections and the same plane group symmetry.     

Mechanism of electroporative dye uptake by mouse B cells.

The color change of electroporated intact immunoglobulin G receptor (Fc gammaR-) mouse B cells (line IIA1.6) after direct electroporative transfer of the dye SERVA blue G (Mr 854) into the cell interior is shown to be dominantly due to diffusion of the dye after the electric field pulse. Hence the dye transport is described by Fick's first law, where, as a novelty, time-integrated flow coefficients are introduced. The chemical-kinetic analysis uses three different pore states (P) in the reaction cascade (C <==> P1 <==> P2 <==> P3), to model the sigmoid kinetics of pore formation as well as the biphasic pore resealing. The rate coefficient for pore formation k(p) is dependent on the external electric field strength E and pulse duration tE. At E = 2.1 kV cm(-1) and tE = 200 micros, k(p) = (2.4 +/- 0.2) x 10(3) s(-1) at T = 293 K; the respective (field-dependent) flow coefficient and permeability coefficient are k(f)0 = (1.0 +/- 0.1) x 10(-2) s(-1) and P0 = 2 cm s(-1), respectively. The maximum value of the fractional surface area of the dye-conductive pores is 0.035 +/- 0.003%, and the maximum pore number is Np = (1.5 +/- 0.1) x 10(5) per average cell. The diffusion coefficient for SERVA blue G, D = 10(-6) cm2 s(-1), is slightly smaller than that of free dye diffusion, indicating transient interaction of the dye with the pore lipids during translocation. The mean radii of the three pore states are r(P1) = 0.7 +/- 0.1 nm, r(P2) = 1.0 +/- 0.1 nm, and r(P3) = 1.2 +/- 0.1 nm, respectively. The resealing rate coefficients are k(-2) = (4.0 +/- 0.5) x 10(-2) s(-1) and k(-3) = (4.5 +/- 0.5) x 10)(-3) s(-1), independent of E. At zero field, the equilibrium constant of the pore states (P) relative to closed membrane states (C) is K(p)0 = [(P)]/[C] = 0.02 +/- 0.002, indicating 2.0 +/- 0.2% water associated with the lipid membrane. Finally, the results of SERVA blue G cell coloring and the new analytical framework may also serve as a guideline for the optimization of the electroporative delivery of drugs that are similar in structure to SERVA blue G, for instance, bleomycin, which has been used successfully in the new discipline of electrochemotherapy.     

The Rubella Virus Nonstructural Protease Requires Divalent Cations for Activity and Functions in trans

The rubella virus (RUB) nonstructural (NS) protease is a papain-like cysteine protease (PCP) located in the NS-protein open reading frame (NSP-ORF) that cleaves the NSP-ORF translation product at a single site to produce two products, P150 (the N-terminal product) and P90 (the C-terminal product). The RUB NS protease was found not to function following translation in vitro in a standard rabbit reticulocyte lysate system, although all of the other viral PCPs do so. However, in the presence of divalent cations such as Zn²⁺, Cd²⁺, and Co²⁺, the RUB NS protease functioned efficiently, indicating that these cations are required either as direct cofactors in catalytic activity or for correct acquisition of three-dimensional conformation of the protease. Since other viral and cell PCPs do not require cations for activity and the RUB NS protease contains a putative zinc binding motif, the latter possibility is more likely. Previous in vivo expression studies of the RUB NS protease failed to demonstrate trans cleavage activity (J.-P. Chen et al., J. Virol. 70:4707–4713, 1996). To study whether trans cleavage could be detected in vitro, a protease catalytic site mutant and a mutant in which the C-terminal 31 amino acids of P90 were deleted were independently introduced into plasmid constructs that express the complete NSP-ORF. Cotranslation of these mutants in vitro yielded both the native and the mutated forms of P90, indicating that the protease present in the mutated construct cleaved the catalytic-site mutant precursor. Thus, RUB NS protease can function in trans.     

Noninvasive oxygen measurements and mass transfer considerations in tissue culture flasks

Murine hybridomas were cultivated in tissue culture flasks. Dissolved oxygen tensions in the gas and liquid phases during cell growth were monitored. Oxygen levels were measured noninvasively by interrogating an oxygen-sensitive patch mounted on the interior surface of the tissue culture flask with an optrode from outside the tissue culture flask. Readings were made in tissue culture flasks with caps both cracked open and completely closed. Although the oxygen in the gas phase remained near atmospheric oxygen levels in both flasks, over time the liquid-phase oxygen tension at the bottom of the flasks reached zero during cell growth in both the open and closed tissue culture flasks. These results suggest that the widespread practice of cracking open tissue culture flask caps during cell growth with a view to supplying adequate oxygen to cells is ineffective and probably unnecessary.The mass transfer characteristics of the tissue culture flask were also studied. The dominant resistance to oxygen mass transfer to the sensor and the cells was through the liquid media. The mass transfer rates through the liquid layer under standard laboratory conditions were found to be greater than those predicted by diffusion alone. This suggests that mixing at a microscale occurs. Volumetric and specific oxygen consumption rates were also calculated from the sensor data. These consumption rates were comparable with values published elsewhere. (c) 1996 John Wiley & Sons, Inc.