Transpiration and drought resistance of Douglas-fir seedlings exposed to excess ammonium

 In a pot trial growth and transpiration of 3-year-old Douglas-fir seedlings on an acid, sandy soil was examined at a deficient (30 kg N ha^{–  1} year^{–  1}) and an excessive level (120 kg N ha^{–  1} year^{–  1}) of NH₄ application. Dissolved ammonium sulphate was applied to the pots weekly for two growing seasons. In half of the pots a complete set of other nutrients was applied in optimal proportions to the applied nitrogen. Water supply was optimal and transpiration was recorded. At the end of the second treatment season irrigation was stopped for 2 weeks during dry and sunny weather. Both high application of NH₄ and additional nutrients increased shoot growth and transpiration demand in the first treatment year. The root system was smaller at higher N level and this reduced water uptake accordingly. In the second year the combination of high NH₄ ⁺ and additional nutrients affected root functioning predominantly due to salinity effects and this seriously decreased water uptake capacity and shoot water potentials, finally resulting in tree death. Without addition of other nutrients the high NH₄ ⁺ application resulted in a high degree of soil acidification, which damaged the roots, that showed a decrease in water uptake capacity. At the low NH₄ supply level soil acidification was lower, and root functioning was not affected, and the trees recovered quickly from the imposed drought. Higher needle K and P status depressed transpiration rates at the low NH₄ application rate. Received: 9 January 1995 / Accepted: 18 September 1995     

Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study.

Time-resolved fluorescence and fluorescence anisotropy data surfaces of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii in 80% glycerol have been obtained by variation of excitation energy and temperature between 203 and 303 K. The fluorescence kinetics of a deletion mutant lacking 14 COOH-terminal amino acids were compared with the wild-type enzyme to study a possible interaction of the COOH-terminal tail with the active site of the enzyme. The flavin adenine dinucleotide fluorescence in both proteins exhibits a bimodal lifetime distribution as recovered by the maximum entropy method of data analysis. The difference in standard enthalpy and entropy of associated conformational substates was retrieved from the fractional contributions of the two lifetime classes. Activation energies of thermal quenching were obtained that confirm that the isoalloxazines in the deletion mutant are solvent accessible in contrast to the wild-type enzyme. Red-edge spectroscopy in conjunction with variation of temperature provides the necessary experimental axes to interpret the fluorescence depolarization in terms of intersubunit energy transfer rather than reorientational dynamics of the flavins. The results can be explained by a compartmental model that describes the anisotropy decay of a binary, inhomogeneously broadened, homoenergy transfer system. By using this model in a global analysis of the fluorescence anisotropy decay surface, the distance between and relative orientation of the two isoalloxazine rings are elucidated. For the wild-type enzyme, this geometrical information is in agreement with crystallographic data of the A. vinelandii enzyme, whereas the mutual orientation of the subunits in the deletion mutant is slightly altered. In addition, the ambiguity in the direction of the emission transition moment in the isoalloxazine ring is solved. The anisotropy decay parameters also provide information on electronic and dipolar relaxational properties of the flavin active site. The local environment of the prosthetic groups in the deletion mutant of the A. vinelandii enzyme is highly inhomogeneous, and a transition from slow to rapid dipolar relaxation is observed over the measured temperature range. In the highly homogeneous active site of the wild-type enzyme, dipolar relaxation is slowed down beyond the time scale of fluorescence emission at any temperature studied. Our results are in favor of a COOH-terminal polypeptide interacting with the active site, thereby shielding the isoalloxazines from the solvent. This biological system forms a very appropriate tool to test the validity of photophysical models describing homoenergy transfer.     

Characterization of polygalacturonase-overproducing Aspergillus niger transformants

Summary Aspergillus niger pyrA co-transformants with multiple copies of the gene (pgaII) encoding the major endopolygalacturonase were characterized in detail. Typically, these transformants produced tenfold or more polygalacturonase from plasmids that had integrated in most cases at ectopic sites, in comparison to the untransformed strain. Some mitotic instability was observed upon application of a positive selection procedure for reversion of the pyrA marker. Analysis of these strains indicated that the most frequent event involved is the excision of part of the array of tandemly integrated plasmids, without scrambling of the plasmids remaining in the chromosome. From promoter deletion analysis it was concluded that the pgaII gene is subject to positive control. The putative positive regulatory protein appears not to be limiting for overexpression of the pgaII gene. Correspondence to: J. Visser     

Life span changes in the presence of alpha-melanocyte-stimulating-hormone-containing cells in the human pituitary.

Since recent circumstantial evidence has suggested possible functions of alpha-MSH in intrauterine growth and labour, the presence of this hormone in the human pituitary was determined by means of the indirect immunofluorescence procedure during development and adulthood. Cross reaction of the antibodies with other peptides was measured after which they were purified by solid phase absorption. Experiments on the rat pituitary showed that staining of alpha-MSH- and ACTH-containing cells could be obtained well until 48 h after death. In the pars distalis the ability of ACTH-containing cells to take up stain increased during the period of post-mortem storage. In the youngest human fetus studied (15 weeks) only alpha-MSH-containing cells were found in the pars intermedia and no ACTH-containing cells were observed. In the other fetal pituitaries a distinct pars intermedia containing more alpha-MSH cells than ACTH cells was found. In the pars distalis of the fetuses more ACTH- than alpha-MSH-containing cells were observed. From birth to 19 years, progressively fewer alpha-MSH containing cells could be detected in the 'zona intermedia' and pars distalis, while in adults only a few such cells were found in either area. Irrespective of age, sex, cause of death or therapy, alpha-MSH-containing cells were found in all pituitaries throughout life. The number of ACTH containing cells gradually increased in the zona intermedia and pars distalis and reached a high adult level in the latter structure. In the pituitaries of seven anencephalics, no alpha-MSH-containing cells were present. The presence of alpha-MSH in the fetal pars intermedia, the change in the ratio of the alpha-MSH/ACTH cells during the course of development, and the absence of alpha-MSH in anencephaly all support the possibility that human fetal pituitary alpha-MSH is involved in both intrauterine growth and fetal adrenal function and thus also in parturition.     

The measurement of subnanosecond fluorescence decay of flavins using time-correlated photon counting and a mode-locked Ar ion laser.

A system is described consisting of a mode-locked Ar ion laser and time-resolved photon-counting electronics. The system is capable of measuring fluorescence lifetimes in the subnanosecond time domain. The Ar ion laser is suitable for the excitation of flavins, since the available laser wavelengths encompass the first absorption band of the yellow chromophore. Due to the high radiation density and the short pulse, both the time and wavelength resolution of the fluorescence of very weakly emitting compounds can be measured. Experiments have been described for flavin models exhibiting single and multiple modes of decay. In these examples lifetimes were determined both from deconvolved decay curves and from direct analysis of the tail of the curve, where no interference of the exciting pulse is encountered. Both determinations showed very good agreement. Due to the highly polarized laser light the decay of the emission anisotropy could be measured directly after the exciting pulse. In principle, fast rotational motions might be detected. An anisotropy measurement conducted with a flavoprotein with a noncovalently attached FAD is presented.     

Binding of radioiodinated propylthiouracil to rat liver microsomal fractions. Stimulation by substrates for iodothyronine 5''-deiodinase.

Previous studies have shown that 2-thiouracil derivatives are uncompetitive inhibitors of iodothyronine 5'-deiodinase activity of rat liver microsomal fraction. Therefore the interaction of radioiodinated 6-propyl-2-thiouracil with rat liver microsomal fraction and the effect of substrate, cofactor and other inhibitors of 5'-deiodinase activity activity were investigated. It was found that micromolar concentrations of, in order of increasing potency, 3,5-diiodotyrosine, thyroxine, 3,3',5'-tri-iodothyronine and 3',5'-di-iodothyronine significantly enhanced binding of 5-[125I]iodo-6-propyl-2-thiouracil to the enzyme preparation. This stimulation was not seen in the presence of 1 mM dithiothreitol, 0.1 mM-6-propyl-2-thiouracil, 0.1 mM-6-propyl-2-thiouracil, 0.1 M-2-mercapto-1-methylimidazole or 1 mM-sodium sulphite. These results support the hypothesis that thiouracil derivatives inhibit 5'-deiodinase activity by forming a mixed disulphide with an intermediate enzyme complex, probably a sulphenyl iodide.     

Signs of stabilisation and stable coexistence

Many empirical studies motivated by an interest in stable coexistence have quantified negative density dependence, negative frequency dependence, or negative plant–soil feedback, but the links between these empirical results and ecological theory are not straightforward. Here, we relate these analyses to theoretical conditions for stabilisation and stable coexistence in classical competition models. By stabilisation, we mean an excess of intraspecific competition relative to interspecific competition that inherently slows or even prevents competitive exclusion. We show that most, though not all, tests demonstrating negative density dependence, negative frequency dependence, and negative plant–soil feedback constitute sufficient conditions for stabilisation of two‐species interactions if applied to data for per capita population growth rates of pairs of species, but none are necessary or sufficient conditions for stable coexistence of two species. Potential inferences are even more limited when communities involve more than two species, and when performance is measured at a single life stage or vital rate. We then discuss two approaches that enable stronger tests for stable coexistence‐invasibility experiments and model parameterisation. The model parameterisation approach can be applied to typical density‐dependence, frequency‐dependence, and plant–soil feedback data sets, and generally enables better links with mechanisms and greater insights, as demonstrated by recent studies.     

A new polymorphism in the type II deiodinase gene is associated with circulating thyroid hormone parameters

Type II deiodinase (D2) is important in the regulation of local thyroid hormone bioactivity in certain tissues. D2 in skeletal muscle may also play a role in serum triiodothyronine (T(3)) production. In this study, we identified a polymorphism in the 5'-UTR of the D2 gene (D2-ORFa-Gly3Asp). We investigated the association of D2-ORFa-Gly3Asp, and of the previously identified D2-Thr92Ala polymorphism, with serum iodothyronine levels. D2-ORFa-Gly3Asp was identified by sequencing the 5'-UTR of 15 randomly selected individuals. Genotypes for D2-ORFa-Gly3Asp were determined in 156 healthy blood donors (age 46.3 +/- 12.2 yr) and 349 ambulant elderly men (age 77.7 +/- 3.5 yr) and related to serum iodothyronine and TSH levels. D2-ORFa-Asp(3) had an allele frequency of 33.9% in blood bank donors and was associated with serum thyroxine (T(4); Gly/Gly vs. Gly/Asp vs. Asp/Asp = 7.06 +/- 0.14 vs. 6.74 +/- 0.15 vs. 6.29 +/- 0.27 microg/dl, P = 0.01), free T(4) (1.22 +/- 0.02 vs. 1.16 +/- 0.02 vs. 1.06 +/- 0.04 ng/dl, P = 0.001), reverse T(3) (P = 0.01), and T(3)/T(4) ratio (P = 0.002) in a dose-dependent manner, but not with serum T(3) (P = 0.59). In elderly men, D2-ORFa-Asp(3) had a similar frequency but was not associated with serum iodothyronine levels. This new polymorphism in the 5'-UTR of D2 is associated with iodothyronine levels in blood donors but not in elderly men. We hypothesize that this might be explained by the decline in skeletal muscle size during aging, resulting in a relative decrease in the contribution of D2 to serum T(3) production.     

The relation between cell proliferation,differentiation and ultrastructural development in rat intestinal epithelium

Summary The ultrastructural development of the principal cells in rat small intestine was studied by morphometric analyses in relation to the exact cell position along crypt and villus. From the bottom to the tip of the crypt, a gradual increase occurred in absolute size of the total cell, the cytoplasm, the terminal web and of nearly all cell organelles. Also, the relative size of the cytoplasm, mitochondria, microvilli and endoplasmic reticulum increased during crypt cell differentiation. No sudden changes in ultrastructure were observed in the so-called critical decision zone, normally located halfway up the crypt where the proliferative activity ceases. At the crypt-villous junction a 1.4–3 fold increase in cell size, cytoplasm, terminal web and of most organelles was noted. Expansion of the proliferative cell compartment over the total length of the crypt as occurs during recovery after a low X-irradiation dose (72 h after 400 R) does not affect the normal development of cellular ultrastructure. These findings are discussed in relation to biochemical and cell kinetic data.