IL-1α and TNFα act synergistically to stimulate production of myeloid colony-stimulating factors by cultured human bone marrow stromal cells and cloned stromal cell strains

Human bone marrow stromal cells repond to stimulation by the monokines IL-1 and TNF by producing colony-stimulating factors such as GM-CSF and G-CSF. In this study we show that IL-1α and TNFα act synergistically to stimulate GM-CSF and G-CSF production by cultured marrow stromal cells. We further show that IL-1α and TNFα synergistically stimulate production of GM-CSF and G-CSF by a clonal stroma-derived cell strain. Although IL-1 and TNF share many of the same biological activities, we show that IL-1α and TNFα have an unequal ability to induce myeloid-CSF production by both cultures, with IL-1α being the more potent inducer. We found that induction by IL-1α and TNFα was independent of cell proliferation. The effect of IL-1α and TNFα on production of the two myeloid-CSFs by the clonal cells was significantly greater than the unfractionated passaged stromal cultures, having the greater effect on G-CSF production. The clonally derived stromal cells constitutively produced colony-stimulating activity, in particular GM-CSF, at levels easily detected by ELISA. These findings show that, in addition to the overlapping and additive activities of IL-1α and TNFα, they can interact synergistically. Our findings further suggest that a small subpopulation of stroma cells may be the major producer of G-CSF in the marrow microenvironment during immune response. © 1994 wiley-Liss, Inc.     

THE GENETICS OF AN ISOLATING MECHANISM BETWEEN TWO SIBLING SPECIES OF DROSOPHILA

Matings between Drosophila simulans females and males of the sibling species D. mauritiana are of abnormally short duration. These rapid matings interrupt the transfer of sperm, leading to substantial reproductive isolation in interspecific as compared to intraspecific copulations. Genetic analysis of this behavior shows that it is influenced much more by the male than the female genotype, with genes from D. simulans being dominant. In males, shortened copulation is caused by interspecific divergence at a minimum of three loci, with one gene on each of the major chromosomes. This is an underestimate of the true number of loci affecting the trait, which could be much larger. The two autosomes have the largest effect, whereas that of the X chromosome is much smaller. The genetic architecture of copulation duration and the larger effect of male than female genotype suggest that females can detect and discriminate against differences in male genitalia.     

Purification and characterization of bovine brain calmodulin-dependent protein kinase II. The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme

Bovine brain contains two calmodulin-dependent phosphodiesterase kinases which are separated on Sephacryl S-300 column. One of these kinases has been purified to homogeneity and shown to belong to the calmodulin-dependent protein kinase II family. Phosphorylation of the 63 kDa phosphodiesterase by this purified protein kinase results in the incorporation of 1.0 mol phosphate per mol subunit and an accompanying increase in Ca²⁺ concentrations required for the phosphodiesterase activation by calmodulin. The protein kinase undergoes autophosphorylation to incorporate 1.0 mol phosphate per mol of subunit of the enzyme and the autophosphorylated enzyme is active, independent of the presence of Ca²⁺. The autophosphorylation reaction as well as the protein kinase reaction are rendered Ca²⁺ independent in less than 15 seconds when approximately one mol phosphate per mol protein kinase is incorporated. The result suggests that activation of phosphodiesterase phosphorylation reaction may occur prior to the activation of phosphodiesterase and phosphatase during a cell Ca²⁺ flux via the protein kinase autophosphorylation mechanism.Abbreviations SDS sodium dodecyl sulfate - EGTA ethylene glycol bis (-aminoethyl ether) - N,N,N,N tetra acetic acid - EDTA ethylenediamine-tetraacetic acid - cAMP cyclic adenosine 35 monophosphate This work is supported by grants from the Medical Research Council of Canada (JHW), the Heart and Stroke Foundation of Alberta (JHW and RKS) and the Heart and Stroke Foundation of Saskatchewan (RKS)     

GERMINATION ECOPHYSIOLOGY OF HERBACEOUS PLANT SPECIES IN A TEMPERATE REGION

Germination phenology data have been collected from 75 winter annuals, 49 summer annuals, 28 monocarpic perennials, and 122 polycarpic perennials, and experimental investigations of dormancy breaking and germination requirements have been conducted on 56 winter annuals, 32 summer annuals, 18 monocarpic perennials, and 73 polycarpic perennials. The purpose of these studies was to determine if there are correlations between the dormancy breaking and germination requirements of seeds and the germination phenology, life cycle type, habitat requirements, range of geographical distribution, and phylogenetic relationships of the species. Germination phenology is highly correlated with the responses of seeds to the yearly temperature cycle. Species with winter and summer annual life cycles have predictable germination characteristics, but monocarpic and polycarpic perennials do not. Several dormancy types may be found in a given habitat, and narrowly endemic and widely-distributed species in the same genus may have similar germination characteristics. Within some families there is a tendency for a particular type of seed-temperature response to be very important, but frequently this is related to the predominance of a given life cycle type in the family.     

Analysis of developmental switching in transgenic mice with 5′ and 3′ deletions in the human β globin gene

Our interest in thecis-acting elements that promote the up-regulation of the globin gene has led to a systematic deletion analysis of portions of the globin gene in the context of the HS2 and globin gene using transgenic mice. In constructs that delete the 5 region to only 265 bp, high-level erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between –122 and –265. In addition, a construct in which the entire globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3 region of the globin gene was deleted in order to examine the effect of a previously defined 3 enhancer region. With deletion of this region, the expression of the human globin gene in transgenic mice is unchanged relative to the parental constructs.     

Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

Oligozymes

The simple use of nonisotopic hybridization probes to detect complementary sequences provides valuable information in a large number of research and commercial applications. In hybridization assays, the four ‘S’s (speed, simplicity, sensitivity, and specificity) are important criteria for determining the choice of probe and label. The direct chemical combination of synthetic oligonucleotide probes and enzyme labels offer advantages unmatched by other approaches, with the oligonucleotide providing rapid hybridization and high specificity, and the direct enzyme label providing simple and sensitive detection. Such oligonucleotide-enzyme conjugates (“oligozymes”) can be used in a variety of hybridization and detection formats, including dot blots, Southern/northern blots,in situ, and solution hybridization/capture schemes. The practical synthesis and use of such oligozymes are summarized.     

Effects of UV-B on flavonoids, ferulic acid, growth and photosynthesis in barley primary leaves

Barley (Hordeum vulgare L.) was grown with UV-B (280–320 nm) at levels simulating 25 nr 5% ozone depletion on the date of the summer solstice al 40°N latitude, with UV-A (320–400 nm), or with no supplemental irradiation. In plant growth chambers providing 300 μmol m^?2 s^?1 photosynthetically active radiation (PAR). UV-B-grown leaves elongated more slowly than controls but reached the same final length 1 day later. Leal specific fresh weight (mass leaf area^?1) was significantly increased by UV-B after the 7th day of growth. IV-B did not significantly affect leaf area, fresh weight, dry weight, total chlorophylls, total carotenoids or photosynthetic quantum efficiency. CO₂ assimilation was decreased by UV-B only at internal CO₂ levels above 250 μl l^?1. By the 8th day of growth, UV-B increased flavonoid (saponarin and lutonarin) accumulation in both the lower epidermis and the mesophyll: about 40% of the saponarin and 20% of the lutonarin were in the lower epidermis under all experimental conditions. Glasshouse conditions proved too variable for reproducible determination of growth and photosynthesis but were reliable for determining developmental changes in flavonoid (saponarin and lutonarin) accumulation and provided up to 800 μmol m^?2 s^?1 PAR. In the glasshouse UV-B-grown leaves had more flavonoids than controls al all stages from 5 to 30 days after planting: ca 509 more saponarin and 100% more lutonarin. Levels of soluble (vacuolar) ferulic acid esters were similar under all conditions on day 5. and on day 20 or later, but were significantly higher in UV-B-grown plants on days 10 and 15. UV-B decreased insoluble (cell-wall-bound) ferulic acid esters on a whole leaf basis but significantly increased this fraction in the lower epidermis. UV-A had no significant effects on growth, photosynthesis or ferulic acid, but it slightly increased flavonoid accumulation. The results are discussed in terms of secondary phenolics as a tissue-specific, developmentally regulated adaptive response to UV-B.