Activation of mast cells by systemic hypoxia, but not by local hypoxia, mediates increased leukocyte-endothelial adherence in cremaster venules.

Systemic hypoxia, produced by lowering inspired Po2, induces a rapid inflammation in several microcirculations, including cremaster muscle. Mast cell activation is a necessary element of this response. Selective reduction of cremaster microvascular Po2 (PmO2) with normal systemic arterial Po2 (PaO2; cremaster hypoxia/systemic normoxia), however, does not elicit increased leukocyte-endothelial adherence (LEA) in cremaster venules. This could be due to a short time of leukocyte exposure to the hypoxic cremaster environment. Conversely, LEA increases when PaO2 is lowered, while cremaster PmO2 remains high (cremaster normoxia/systemic hypoxia). An alternative explanation of these results is that a mediator released from a central site during systemic hypoxia initiates the inflammatory cascade. We hypothesized that if this is the case, cremaster mast cells would be activated during cremaster normoxia/systemic hypoxia, but not during cremaster hypoxia/systemic normoxia. The microcirculation of rat cremaster muscles was visualized by using intravital microscopy. Cremaster PmO2 was measured with a phosphorescence quenching method. Cremaster hypoxia/systemic normoxia (PmO2 7 +/- 1 Torr, PaO2 87 +/- 2 Torr) did not increase LEA; however, topical application of the mast cell activator compound 48/80 under these conditions did increase LEA. The effect of compound 48/80 on LEA was blocked by topical cromolyn, a mast cell stabilizer. LEA increased during cremaster normoxia/systemic hypoxia, (PmO2 64 +/- 5 Torr, PaO2 33 +/- 2 Torr); this increase was blocked by topical cromolyn. The results suggest that mast cell stimulation occurs only when PaO2 is reduced, independent of cremaster PmO2, and support the idea of a mediator that is released during systemic hypoxia and initiates the inflammatory cascade.     

Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems

Nicotiana plumbaginifolia protoplasts were directly transformed by PEG treatment with a cloned 16S rRNA gene isolated from a double antibiotic-resistant Nicotiana tabacum plastid mutant. Putative plastid transformants were selected in cell culture by their spectinomycin resistance and identified by their unselected streptomycin resistance. Alternatively, cell lines were selected in the presence of both antibiotics. The cell line (and its regenerated plants) selected solely for spectinomycin resistance demonstrated an extensive segregation of streptomycin resistance in subsequent tests, while the double-selected line showed stable resistance for both antibiotics. The resistance markers were inherited maternally. In the putative plastid transformants the origin of the resistance mutations was identified by the absence of an Aat ll site, missing in the donor N. tabacum plastid gene (spectinomycin resistance site) but present in that of wild-type N. plumbaginifolia , and a sequence analysis of the particular nucleotide changes in both resistance sites. Restriction enzyme analysis of total plastid DNA (ptDNA), and the recloning and full sequencing of the fragment introduced, investigated in one of the plastid transformants, showed no DNA rearrangements accompanied with the integration process. Sequence analysis indicated a targeted, homologous integration of the DNA fragment introduced but an unexpectedly complete homology of the parental ptDNA sequences in this region prevented the location of borders. Although the frequency of plastid transformant colonies (2 × 10⁻⁵) should still be improved, this method for stable chloroplast DNA transformation is comparable with or more efficient than the particle bombardment techniques.     

A streptomycin-resistant line of Nicotiana sylvestris unable to flower

Summary A streptomycin-resistant cell line, SR155, was selected in a callus culture initiated from diploid Nicotiana sylvestris.Regenerates of the SR155 line have a grossly altered morphology, an altered peroxidase isoenzyme pattern, and do not flower. The SR155 plants are diploid, but in the karyotype a chromosome translocation was detected. Present address: Department of Genetics, University of Newcastle, Newcastle-upon-Tyne NE1 7RU, U.K.     

Molecular motion of small nonelectrolyte molecules in lecithin bilayers

Summary We have used magnetic resonance spectroscopy, both ESR and¹³C spin relaxation, to measure translational and rotational mobilities and partition coefficients of small nitroxide solutes in dipalmitoyl lecithin liposomes. Above the bilayer transition temperature,T _c, the bilayer interior is quite fluid, as determined from the solutes' rapid rotational and moderately rapid translational motion; the rotational and translational viscosities within the bilayer are _R <1cP and =6–10cP, respectively. and _R are independent of molecular size for all solutes studied, but all were small compared to the size of the phospholipids. , and probably _R , are relatively independent of temperature aboveT _c, but both increase very sharply as temperature is lowered belowT _c; at 32°C, _R increases to 6cP and is greater than 1000 cP. Anisotropy of rotational motion increases gradually as temperature is lowered toT _c, and changes little belowT _c; anisotropy of translational motion was not investigated.¹³C nuclear spin relaxation measurements indicate that translational motion of nitroxide solutes is more rapid in the center of the bilayer than near the polar interface. It takes at least 100 nsec for a solute molecule to cross the bilayer/water interface. We estimate a lower limit of 2 sec/cm for the interfacial resistance to solute diffusion; this result indicates that interfacial resistance dominates permeation across the membrane. The relative solubility, or partition coefficient, is a strong function of solute structure, and decreases abruptly on cooling through the transition temperature. From the partition coefficient and its temperature dependence we calculate the free energy, enthalpy, and entropy of partition. Effects of cholesterol on partition and diffusion coefficients are compatible with the interpretation that bilayers containing cholesterol consist of two phases.     

Leukemia cell lysis by sulphydryl reagents and thiols.

Iodoacetate and other sulfhydryl reagents as well as cysteine and other thiols caused lysis of the mouse leukemic lymphoblasts L5178Y, L1210, and P388 in culture. Lysis by either iodoacetate or cysteine was preceded by ATP depletion. However ATP depletion was not a sufficient explanation for lysis since deoxyglucose depleted ATP to the same extent as did iodoacetate but did not cause lysis. It was concluded that sulfhydryl-disulfide equilibrium was essential to the maintenance of cellular integrity and ATP concentration.