Application of the micropipette technique to the measurement of cultured porcine aortic endothelial cell viscoelastic properties

The viscoelastic deformation of porcine aortic endothelial cells grown under static culture conditions was measured using the micropipette technique. Experiments were conducted both for control cells (mechanically or trypsin detached from the substrate) and for cells in which cytoskeletal elements were disrupted by cytochalasin B or colchicine. The time course of the aspirated length into the pipette was measured after applying a stepwise increase in aspiration pressure. To analyze the data, a standard linear viscoelastic half-space model of the endothelial cell was used. The aspirated length was expressed as an exponential function of time. The actin microfilaments were found to be the major cytoskeletal component determining the viscoelastic response of endothelial cells grown in static culture.     

Tumor necrosis factor's effects on lung mechanics, gas exchange, and airway reactivity in sheep

The macrophage- and monocyte-produced cytokine tumor necrosis factor alpha (TNF alpha) has been proposed as a major mediator of endotoxin-induced injury. To determine if TNF alpha could reproduce the effects of endotoxin on the lung, we intravenously administered 10 micrograms/kg of human recombinant TNF alpha into five chronically instrumented unanesthetized sheep on two occasions to characterize the TNF alpha response and its reproducibility. We assessed changes in lung mechanics, pulmonary and systemic hemodynamics, gas exchange, and the number and type of peripheral blood leukocytes. We also determined airway reactivity by use of aerosolized histamine before and after TNF alpha infusion. Pulmonary arterial pressure (Ppa) peaked within 30 min of initiating the TNF alpha infusion [47.7 +/- 2.2 vs. 15.9 +/- 0.4 (SE) cmH2O at base line] and then returned toward base line over 4 h. There was a brief decline in left atrial pressure after TNF alpha. Pulmonary hypertension was accompanied by leukopenia, neutropenia, and increases in the alveolar-arterial O2 difference (AaDO2). Dynamic lung compliance (Cdyn) declined after TNF alpha, reaching a nadir within 15 min of the initiation of the TNF alpha infusion [0.045 +/- 0.007 vs. 0.093 +/- 0.007 (+/- SE) l/cmH2O at base line]. Resistance to airflow across the lung (RL) increased from 1.2 +/- 0.2 cmH2O.l-1.s at base line, peaking at 5.4 +/- 1.3 cmH2O.l-1.s 30 min after the start of the TNF alpha infusion. Alterations in Cdyn and RL persisted for 4 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of live offspring with predicted genotypes using PCR-RFLP analysis of polar bodies from mouse oocytes

Accurate identification of genotypes in gametes and early embryos could facilitate the efficient production of offspring with desirable traits. This study demonstrates the feasibility of producing offspring with predictable genotypes from micromanipulated mouse oocytes. The Polymerase Chain Reaction (PCR) was used to amplify genes in the IA subregion of the major histocompatibility complex of the mouse. The validity of the approach was demonstrated in experiment 1 with IA haplo-types of unfertilized mouse ova amplified via PCR and distinguished by restriction fragment length polymorphism (RFLP) analysis. In experiment 2, fertilized oocytes were micromanipulated to remove the first and second polar bodies, which were then genotyped by validated PCR-RFLP procedures. Primary oocytes of heterozygous females contain two copies of each of the different alleles. Following meiosis I and II, the genotype of the ovum was predicted by subtracting the alleles observed in micromanipulated polar body samples. Sixty-two fertilized ova were micromanipulated and transferred to recipient females resulting in 27 live offspring (44%). The correct maternal contribution to the embryonic genotype was predicted in 19 of 27 (71%) offspring as confirmed by PCR-RFLP analysis of DNA from pup tails. Predicted genotypes of two pups were not confirmed (7%), whereas no prediction could be made in six cases (22%). © 1995 Wiley-Liss, Inc.     

Lipid synthesis in mycobacteria: characterization of the biotin carboxyl carrier protein genes from Mycobacterium leprae and M. tuberculosis.

The causative agents of leprosy and tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis, have a lipid-rich cell envelope which contributes to virulence and antibiotic resistance. Acyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis, consists in mycobacteria of two subunits, one of which is biotinylated. Genes from M. leprae and M. tuberculosis encoding a biotinylated protein have been cloned and sequenced. Analysis of the derived protein sequences demonstrated the presence of biotin-binding sites and putative ATP-bicarbonate interactions sites, consistent with the proteins having a biotin carboxylase function as well as their being biotin carrier proteins.     

On protein solubility in organic solvent

Solubility of a model protein, hen egg-white lysozyme, was investigated in a wide range of neat nonaqueous solvents and binary mixtures thereof. All solvents that are protic, very hydrophilic, and polar readily dissolve more than 10 mg/mL of lysozyme (lyophilized from aqueous solution of pH 6.0). Only a marginal correlation was found between the lysozyme solubility in a non-aqueous solvent and the letter's dielectric constant or Hildebrand solubility parameter, and no correlation was observed with the dipole moment. Lysozyme dissolved in dimethyl sulfoxide (DMSO) could be precipitated by adding protein nondissolving co-solvents, although the enzyme had a tendency to form supersaturated solutions in such mixtures. The solubility of lysozyme, both in an individual solvent (1,5-pentanediol) and in binary solvent mixtures (DMSO/acetonitrile), markedly increased when the pH of the enzyme aqueous solution prior to lyophilization was moved away from the proteins's isoelectric point. (c) 1994 John Wiley & Sons, Inc.     

Inheritance of restriction fragment length polymorphisms and random amplified polymorphic DNAs in coastal Douglas-fir

A total of 225 new genetic loci [151 restriction fragment length polymorphisms (RFLP) and 74 random amplified polymorphic DNAs (RAPD)] in coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] have been identified using a three-generation outbred pedigree. The Mendelian inheritance of 16 RFLP loci and 29 RAPD loci was demonstrated based on single-locus segregation in a sample of F₂ progeny. One RFLP locus, PtIFG2025, showed segregation distortion. Probe pPtIFG2025 is a loblolly pine cDNA probe encoding for rbcS. The 16 RFLP loci and 23 allozyme loci were also assayed in a sample of 16 Douglas-fir seed-orchard clones. Allelism was determined at 11 of the 16 RFLP loci. RFLPs were able to detect slightly more variation (4.0 alleles per locus) than allozymes (3.1 alleles per locus). The inheritance of an additional 80 RAPD loci was determined based on haploid segregation analysis of megagametophytes from parent tree 013-1. Once 200–300 markers are identified and placed on a genetic map, quantitative trait loci affecting bud phenology will be mapped.     

Effect of various metal ions on growth of two wheat lines known to differ in aluminium tolerance

The effects of aluminium (Al), manganese (Mn), zinc (Zn), copper (Cu), boron (B), iron (Fe), gallium (Ga), scandium (Sc) and lanthanum (La) on growth of an Al-tolerant and an Al-sensitive line of wheat (Triticum aestivum L.) were measured in solution culture. The concentrations of nutrients in the basal nutrient solution were (M) 500 Ca, 100 Mg, 300 K, 600 N (150 NH₄, 450 NO₃), 600 SO₄, 2.5 P, 3 B, 2.5 Fe, 0.5 Zn, 0.5 Mn, 0.1 Cu at a pH of 4.7. The major solution nutrient concentrations were maintained at the nominal concentration with monitoring, frequent additions and weekly renewal. Differentiation in yield between the Al-tolerant and Al-sensitive line only occurred in the presence of Al indicating that, in the long term, none of the other metals tested could be used as an analog for Al. The visual symptoms in the roots of Cu toxicity (in both lines) and Al toxicity (in the sensitive line) were similar. The solution concentration (M) at which yield of the roots of the tolerant line was reduced by 50% was, in order of increasing tolerance, Cu 0.5, Sc 1.1, La 7.1, Ga 8.6, Al 15, Zn 19, Fe 84, B 490 and Mn 600.     

A model of the sodium dependence of dopamine uptake in rat striatal synaptosomes

Initial velocity of uptake of dopamine (DA) has been measured in rat striatal synaptosomes as a function of both [DA] and [Na]. Carrier mediated uptake is totally dependent on external sodium. The data were fitted to a rapid equilibrium model which has been found in previous studies to fit, with appropriate simplification, uptake data for glutamate, GABA, and choline in several brain regions under varying conditions. This model also gives a good fit to the dopamine data. The minimal best fit simplification of this model allows for DA uptake along with two sodium ions and predicts that apparent maximal velocity of uptake should increase with [Na], while the Michaelis-Menten constant should decrease. The minimal best fit model for DA, and a number of kinetic parameters which quantitate the model, are compared to those for the GABA, glutamate, and choline transporters. The results are consistent with a symmetrical, rapid equilibrium model, which has been presented previously for other neurotransmitters and precursors (18). This model offers a unifying basis for understanding the sodium and membrane potential dependence of neurotransmitter transport and the possible participation of transporters in depolarization induced release throughout the CNS.     

Botanical diversity in British mires

Although mires are sometimes regarded as a single, specific and even esoteric habitat-type, in their full range of development-calcareous fens to acidic bogs, lowland swamps to upland flushes, woodlands, sedgelands and mosslands-they occupy a wide range of environmental conditions and sustain a rich botanical resource. Here an outline is given of the types of mires that are widespread in Britain and their botanical resource. It is discussed with reference to some of the environmental variables and management conditions that have been shown to influence the species composition and diversity of their vegetation. Some implications that this has for the classification of mires are outlined.     

EVIDENCE OF AN ORGANIC MATRIX FROM DIATOM BIOSILICA1

Most biominerals appear to be composites of organic material and mineral. Whether biosilica is such a composite is unresolved because of a lack of evidence for such organic components. We present evidence that organic material exists within diatom biosilica and can be extracted using HF/NH₄F solutions from frustules isolated from Cyclotella meneghiniana Kütz and diatomaceous earth. To eliminate organic casing on the silicified frustules as a source of organic materials, the casing was removed by oxidation of frustules with NaOCl before extraction. The removal of the casing was confirmed in that oxidized frustules no longer displayed the ability to be stained with ruthenium red and fluorescamine. Frustules examined with EDXA showed an emission peak from sulfur before treatment but no peak following treatment, indicating that oxidation removed organic sulfur. The organic material obtained from extracts of fresh frustules contained both soluble and insoluble components. Only soluble material was evident in extracts from diatomaceous earth. The soluble material appears to contain glycoproteins with relatively high levels of serine and glycine. The soluble proteins from fresh frustules also appear to be phosphorylated. Indirect evidence is presented that suggests the soluble proteins may contain regions of primary structure enriched in anionic amino acids. The soluble extracts differ from general cell contents when the two fractions are compared, suggesting that frustules contain specialized organic material. The identification of silica-specific organic material suggests that mineralization in diatoms may be in part matrix-mediated.