An Age-Wise Comparison of Human Airway Smooth Muscle Proliferative Capacity

We compared the proliferation of neonatal and adult airway smooth muscle cells (ASMC) with no/moderate lung disease, in glucose- (energy production by glycolysis) or glucose-free medium (ATP production from mitochondrial oxidative phosphorylations only), in response to 10% fetal calf serum (FCS) and PDGF-AA. In the presence of glucose, cell counts were significantly greater in neonatal vs. adult ASMC. Similarly, neonatal ASMC DNA synthesis in 10% FCS and PDGF-AA, and [Ca²⁺]i responses in the presence of histamine were significantly enhanced vs. adults. In glucose-free medium, cell proliferation was preserved in neonatal cells, unlike in adult cells, with concomitant increased porin (an indicator of mitochondrial activity) protein expression. Compared to adults, stimulated neonatal human ASMC are in a rapid and robust proliferative phase and have the capacity to respond disproportionately under abnormal environmental conditions, through increased mitochondrial biogenesis and altered calcium homeostasis.     

Changing water use and adaptive strategies along rainfall gradients in Mediterranean lupins

• There is growing interest in harnessing the genetic and adaptive diversity of crop wild relatives to improve drought resilience of elite cultivars. Rainfall gradients exert strong selection pressure on both natural and agricultural ecosystems. Understanding plant responses to these facilitates crop improvement.
• Wild and domesticated narrow‐leafed lupin (NLL) collected along Mediterranean terminal drought stress gradients was evaluated under contrasting reproductive phase water supply in controlled field, glasshouse and cabinet studies. Plant phenology, growth and productivity, water use and stress responses were measured over time.
• There is an integrated suite of adaptive changes along rainfall gradients in NLL. Low rainfall ecotypes flower earlier, accumulate lower seed numbers, biomass and leaf area, and have larger root:shoot ratios than high rainfall ecotypes. Water‐use is lower and stress onset slower in low compared to high rainfall ecotypes. Water‐use rates and ecotypic differences in stress response (Ψleaf decline, leaf loss) are lower in NLL than yellow lupin (YL). To mitigate the effects of profligate water use, high rainfall YL ecotypes maintain higher leaf water content over declining leaf water potential than low rainfall ecotypes. There is no evidence for such specific adaptation in NLL.
• The data suggests that appropriate phenology is the key adaptive trait to rainfall gradients in NLL because of the flow‐on effects on biomass production, fitness, transpiration and stress onset, and the lack of physiological adaptations as in YL. Accordingly, it is essential to match phenology with target environment in order to minimize risk and maximize yield potential.

     

Genetic instability of industrial strains of Penicillium chrysogenum

Summary It has shown that several characteristics of high-producing industrial strains of Penicillium chrysogenum tend to segregate in the course of cultivation (slant-to-slant transfer). Segregation includes a decrease in the yield of penicillin, mean conidial size, mean size of the nuclei, and an increase in the proportion of morphologically wild-type colonies. These lower-producing segregants also have a higher sensitivity against ultraviolet radiation and, as shown by cytofluorometric methods, a lower DNA content in the condia, a decrease in phosphate uptkae and in the activity of extracellular alkaline phosphatases compared to high-producing strains. Obviously, during mutagenesis/selection programmes ploidy mutants have been selected, which entails an increase in the number of genes coding enzymes responsible for penicillin biosynthesis. In the absence of selection pressure these high-producing strains segregate to lower-producing strains by chromosome losses in the course of slant-to-slant transfers. Offprint requests to: W. Künkel     

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.     

A quantitative study on recovery from acute haemorrhage in adult laboratory rats. II. Bone marrow, spleen and liver morphology

We investigate bone marrow, spleen and liver morphology in adult laboratory rats following acute haemorrhage to establish their validity in preclinical studies, especially in safety drug evaluations. The most marked changes were found in the proportion of spleen erythroid nucleated cells. Higher E: M ratios and relative erythroid cell counts were also marked in the bone marrow. Their increase was not so intensive and disappeared earlier than the increase of spleen erythropoiesis. Variations in counts of marrow neutrophils and their precursors were not specific.     

Thermodynamic characterization of daunomycin-DNA interactions: microcalorimetric measurements of daunomycin-DNA binding enthalpies

We report the first direct determination of binding enthalpies for the complexation of monomeric daunomycin with a series of 10 polymeric DNA duplexes. These measurements were accomplished by using a recently developed stopped-flow microcalorimeter capable of detecting reaction heats on the microjoule level. This enhanced sensitivity allowed us to measure daunomycin-DNA binding enthalpies at monomeric drug concentrations (e.g., 10-20 microM), thereby precluding the need to correct for daunomycin self-association, as has been required in previous batch calorimetric studies [Remeta, D. P., Marky, L. A., & Breslauer, K. J. (1984) Abstracts of Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy, 838a; Breslauer, K. J., Remeta, D. P., Chou, W. Y., Ferrante, R., Curry, J., Zaunczkowski, D., Snyder, J. G., & Marky, L. A. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8922-8926]. We correct the published daunomycin-DNA binding enthalpies measured by batch calorimetry at higher drug concentrations (e.g., 0.5-1.0 M) for the enthalpy contribution associated with the binding-induced disruption of drug aggregates. The requisite correction term was obtained from a van't Hoff analysis of temperature-dependent NMR measurements on daunomycin solutions. We find remarkable agreement between the net binding enthalpies derived from these corrected batch calorimetric data and the corresponding binding enthalpies measured directly by stopped-flow microcalorimetry. The enhanced sensitivity of the stopped-flow instrument also allowed us to evaluate the influence of drug binding density on the daunomycin-DNA binding enthalpies. This assessment was accomplished by conducting stopped-flow calorimetric measurements over a range of seven different drug-to-phosphate ratios (r). For most of the 10 DNA host duplexes studied, we find that the daunomycin binding enthalpies exhibit small but significant r dependencies. The sensitivity of the stopped-flow instrument also enabled us to detect significant dilution enthalpies for several of the drug-free DNA duplexes, a quantity generally assumed to be negligible in previous studies. We discuss the binding enthalpies, their dependence on binding density, and the duplex dilution enthalpies in terms of the influence of base composition, sequence, conformation/hydration, and binding cooperativity on the sign and the magnitudes of the daunomycin-DNA binding enthalpy data reported here.     

Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.