A morphometric study quantifying the mucus content of proliferating colonic epithelial cells.

We report a quantification of the maximum mucus accumulation in proliferating rat colonic epithelial cells. The proliferative potential was determined by radioautographic study of one-hour pulse exposures to tritiated thymidine, mucous content was determined by Periodic-acid Schiff (PAS) staining. We examined 55 labeled mucous cells in 0.5- to 1-micrometer serial sections. The maximum thecal and nuclear profiles of these cells were photographed and their surface areas were determined utilizing a coordinate sensor. The data were expressed as a theca-to-nucleus (T/N) ratio. The maximum (T/N) ratio for a labeled mucous cell was 3.0. We performed a similar analysis on 22 unlabeled mucous cells from upper crypt regions and surface epithelium to derive the range of (T/N) ratios for terminally differentiated mature mucous cells. The range of (T/N) ratios from these cells was from 4.8 to 16.4. Our study shows that proliferative potential of mucous cells is determined by the interrelationship between mucus accumulation and nuclear size.     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

Solution pH modifies the response of Norway spruce seedlings to aluminium

Norway spruce (Picea abies) was exposed to nutrient solutions containing a range of aluminium (Al) concentrations at several pH levels (3.2, 4 and 5). Root growth was reduced by 100 µM and 400 µM Al at pH 4 and 5, but at pH 3.2 only by 400 µM Al. The Al content of the roots increased with increasing pH. The Al content of the roots was higher at the root tips than at the older root parts at all pH values. Using X-ray microanalysis it could be shown that higher levels of Al at increased pH were mainly due to increased Al contents in root cortex cell walls. In seedlings, mycorrhizal with Pisolithus tinctorius or Lactarius rufus, the Al concentration of cortex cell walls was higher when nitrate (NO₃) rather than ammonium (NH₄) was the nitrogen (N) source.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

NH4 : NO3 nutrition influence on biomass productivity and root respiration of poplar and willow clones

Nitrogen fertilization often improves the yield of intensively managed, short‐rotation coppices. However, information of N nutrition form on the growth of common species and clones used for biomass production is limited. Thus, this study aims at evaluating N form effects on the growth of two Salicaceae clones. Cuttings of the poplar clone Max 4 (Populus maximovizcii × P. nigra) and the willow clone Inger (Salix triandra × S. viminialis) were fertilized in a pot experiment with four ratios of nitrate (NO₃^?) to ammonium (50%, 62.5%, 75% and 87.5% NO₃^? balanced with ammonium (NH₄⁺) to constant total N) for one growing season and under stable soil pH. Plants were harvested for analysis of biomass and morphology of leaves, stem and roots. Respiration of fine and coarse roots (RR) was determined and related to biomass growth. Salix cv. Inger accumulated more total dry matter than Populus cv. Max 4. In both Salicaceae clones, the total biomass was significantly influenced by the nitrate ratio and greatest in plants fertilized with 50% NO₃^? of the total N supply. Both clones possess a different leaf and root morphology, but no significant influence of the NO₃^? ratio on the morphology was found. Fine RR rates differed significantly between clones, with significantly greater fine RR in Max 4; 87.5% NO₃^? fertilization increased the fine RR. Fine RR and total accumulated plant biomass were closely related. Our study is the first to show the tremendous influence of fine root respiration, especially including the carbon‐intensive reduction of NO₃^? to NH₄⁺, on the aboveground growth of Salicaceae clones. Ways to improve yield in SRC are thus to lower the assimilate consumption by fine roots and to match fertilization regimes to the used clones or vice versa.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

Fine root biomass and turnover in southern taiga estimated by root inclusion nets

Fine roots play an important part in forest carbon, nutrient and water cycles. The turnover of fine roots constitutes a major carbon input to soils. Estimation of fine root turnover is difficult, labour intensive and is often compounded by artefacts created by soil disturbance. In this work, an alternative approach of using inclusion nets installed in an undisturbed soil profile was used to measure fine root production and was compared to the in-growth core method. There was no difference between fine root production estimated by the two methods in three southern taiga sites with contrasting soil conditions and tree species compositions in the Central Forest State Biosphere Reserve, Russia. Expressed as annual production over standing biomass, Norway spruce fine root turnover was in the region of 0.10 to 0.24 y^-1. The inclusion net technique is suitable for field based assessment of fine root production. There are several advantages over the in-growth core method, due to non-disturbance of the soil profile and its potential for very high rate of replication.     

Cadmium-induced changes in antioxidative systems, hydrogen peroxide content, and differentiation in Scots pine roots.

To investigate whether Cd induces common plant defense pathways or unspecific necrosis, the temporal sequence of physiological reactions, including hydrogen peroxide (H(2)O(2)) production, changes in ascorbate-glutathione-related antioxidant systems, secondary metabolism (peroxidases, phenolics, and lignification), and developmental changes, was characterized in roots of hydroponically grown Scots pine (Pinus sylvestris) seedlings. Cd (50 microM, 6 h) initially increased superoxide dismutase, inhibited the systems involved in H(2)O(2) removal (glutathione/glutathione reductase, catalase [CAT], and ascorbate peroxidase [APX]), and caused H(2)O(2) accumulation. Elongation of the roots was completely inhibited within 12 h. After 24 h, glutathione reductase activities recovered to control levels; APX and CAT were stimulated by factors of 5.5 and 1.5. Cell death was increased. After 48 h, nonspecific peroxidases and lignification were increased, and APX and CAT activities were decreased. Histochemical analysis showed that soluble phenolics accumulated in the cytosol of Cd-treated roots but lignification was confined to newly formed protoxylem elements, which were found in the region of the root tip that normally constitutes the elongation zone. Roots exposed to 5 microM Cd showed less pronounced responses and only a small decrease in the elongation rate. These results suggest that in cells challenged by Cd at concentrations exceeding the detoxification capacity, H(2)O(2) accumulated because of an imbalance of redox systems. This, in turn, may have triggered the developmental program leading to xylogenesis. In conclusion, Cd did not cause necrotic injury in root tips but appeared to expedite differentiation, thus leading to accelerated aging.