Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.     

Reactivity and stability of mycelium-bound carboxylesterase from Aspergillus oryzae.

The reactivity and thermostability of a novel mycelium-bound carboxylesterase from lyophilized cells of Aspergillus oryzae are explored in organic solvent. Ethanol acetylation was selected as reference esterification reaction. High carboxylesterase activity cells were used as biocatalyst in batch esterification tests at 12.5 < S(o) < 125 mmol L(-1), 5.0 < X(o) < 30 g L(-1), 0.49 < log P < 4.5 and 30 < T < 80 degrees C, as well as in residual activity tests after incubation at 40 < T < 90 degrees C. The starting rates of product formation were used to estimate with the Arrhenius model the apparent activation enthalpies of the enzymatic reaction (29-33 kJ mol(-1)), the reversible unfolding (56-63 kJ mol(-1)), and the irreversible denaturation (22 kJ mol(-1)) of the biocatalyst.     

Synthesis and structure of [bis(pyrazol-1-yl)acetato]trioxorhenium(VII) and [bis(3,5-dimethylpyrazol-1-yl)acetato]trioxorhenium(VII)

The coordination of the ligands bis(pyrazol-1-yl)acetate (bpza) and bis(3,5-dimethylpyrazol-1-yl)acetate (bdmpza) to rhenium(VII) was investigated. The compounds [(bpza)ReO₃] and [(bdmpza)ReO₃] were synthesised by reaction of bpza and bdmpza with perrhenic acid with the loss of one water molecule. The new complex [(bdmpza)ReO₃] was characterised by single-crystal X-ray analysis. It has a monomeric structure with a distorted octahedron for the [N,N,O]ReO₃ central core.     

Effects of repeated (NH4)2SO4 application on sulfur pools in soil,soil microbial biomass,and ground vegetation of two watersheds in the Black Forest/Germany

The effect of repeated (NH₄)₂SO₄ applications (3 × 700 kg ha^–1 in 1988, 1991, and 1994, respectively) on S pools in soil, soil microbial biomass, and ground vegetation was studied at two Norway spruce (Picea abies L. [Karst.]) sites in the Black Forest/Germany. In both eco-systems, most of the total S pool was located in the soil. The soil also was the predominant compartment for retention of applied SO₄ ^2--S. The fractions of organic and inorganic S forms in the initial soil S content, and the retention of experimentally applied S was different for both sites. In the podzol Schluchsee, organic S accounted for 92% of total S. In the cambisol Villingen, the S pool consisted of 33% organic S and 67% inorganic S. The retention of applied S in various compartments of both ecosystems reflected these proportions. Only minor amounts of fertilized S (<1%) was retained in the spruce trees, ground vegetation, and soil microbial biomass. However, between 51% (Villingen) and 72% (Schluchsee) of the applied S was retained in the soil. In the Schluchsee podzol, 75% of retained fertilizer S was accumulated as ester sulfate, whereas SO₄ ^2-adsorption and precipitation of Al hydroxy sulfates were restricted by dissolved organic matter in the soil solution. In the Villingen cambisol, SO₄ ^2- adsorption was the dominant process of S retention, although 20% of the fertilized S again was retained as ester sulfate. The significant relevance of organic S forms in the retention of fertilizer S in both soils emphasizes the need for models which include the formation and re-mineralization of organic S compounds, especially of ester sulfates, for correctly simulating and predicting the retention and remobilization of S in acid forest soils subject to changing atmospheric N and S deposition.     

Uptake of 15N fertilizer in compost-amended soils

Composts are considered low analysis fertilizers because their nitrogen and phosphorus content are around 1% and the organic nitrogen mineralization rate is near 10%. If compost is added to agricultural land at the N requirement of grain crops (40 – 100 kg N ha^–1), application rates approach 40–100 mg ha^–1. Much lower rates may be advisable to avoid rapid accumulation of growth limiting constituents such as heavy metals found in some composts. Combining low amendment rates of composts with sufficient fertilizer to meet crop requirements is an appealing alternative which (a) utilizes composts at lower rates than those needed to supply all the crop N requirement, (b) reduces the amount of inorganic fertilizer applied to soils, and (c) reduces the accumulation of non-nutrient compost constituents in soils. A study was conducted to compare the effects of blends of biosolids compost (C) with ¹⁵N urea(U) or ¹⁵NH4 ¹⁵NO3 (N) fertilizers to fertilizer alone on tall fescue (Festuca arundinacea L.) growth and N uptake. Blends which provided 0, 20, 40 or 60 mg N kg^–1 application rate as compost N and 120, 100, 80 or 60 mg N kg^–1 as fertilizer N, respectively, were added to Sassafras soil (Typic Hapludults). Fescue was grown on the blends in a growth chamber for 98 days. Fescue yields recorded by clippings taken at 23, 46 and 98 days and roots harvested after the 98-day clipping increased with increasing fertilizer level for both NH₄NO₃ and urea and with or without compost. Nitrogen uptake by fescue responded similarly to yield with increases recorded with increasing fertilizer levels with or without compost. Paired comparisons based on cumulative 98-day clippings data showed that yields from blends were equal to yields from fertilizer treatments containing the same percentage of fertilizer as the blends. These data indicated that compost did not provide sufficient plant-available N to increase yields or N uptake. None of the blends equaled 120 mg N kg^–1 fertilizer rate except for 100 mg NH₄NO₃-or urea-N kg^–1 –20 mg compost-N kg^–1blends. The data suggest that biosolids compost blended with fertilizer at a rate of 2–6 mg ha ^–1 did not supply sufficient additional available N to increase yields or N uptake over those of fertilizer alone.     

The importance of root carbohydrate abundance in ammonium uptake

The influence of carbohydrates on ammonium uptake and ammonium transporter (AMT1) expression was investigated in roots of field pea (Pisum arvense) and rutabaga (Brassica napus var. rapifera). Ammonium transport into field pea seedlings diminished markedly following cotyledon removal, which indicated that uptake of ammonium was under control of reserves stored in the cotyledons. Excision of cotyledons decreased also the level of some amino acids, glucose and total reducing sugars in field pea roots. To investigate the importance of the sugar supply for the regulation of ammonium uptake at low external NH ₄ ⁺ level, 1 mM glucose or sucrose was supplied for several hours to the field pea seedlings deprived cotyledons or to intact rutabaga plants. Supply of both sugars resulted in a substantial increase in ammonium uptake by both plant species and enhanced markedly the expression of AMT1 in rutabaga roots. The results indicate that sugars may regulate ammonium transport at the genetic level.     

Diurnal changes in the concentrations of 2-phenylethyl β-D-glucopyranoside and the corresponding volatile aglycone in the tissue and headspace of Trifolium repens L. florets

Distinct synchronous diurnal rhythms were detected in the concentrations of phenethyl alcohol and phenethyl acetate in the tissue of blooming Trifolium repens florets. Corresponding rhythmic oscillations were observed for the same two compounds in the floral headspace. Maximum content of the volatiles in the tissue and headspace was observed 3–9 h after initiation of the photophase. The concentrations of phenethyl alcohol and phenethyl acetate in the tissue increased significantly during floral development. At full bloom the tissue contained amounts sufficient to support 2–3 h of emission. Several observations suggested that esterification of phenethyl alcohol was the source for phenethyl acetate. Trimethylsilyl derivatization and enzymatic hydrolysis of fractionated flower extracts identified 2‐phenylethyl β‐d ‐glucopyranoside as the major glucoside in the florets. The pool of glucosides increased significantly during floral development and at full bloom 97% of the phenethyl alcohol was bound as glucoside. The concentration of 2‐phenylethyl β‐d ‐glucopyranoside did not vary in a rhythmic diurnal manner. The dynamics among the diurnal rhythmic phenomena in the tissue and headspace and the fraction of volatiles bound as glucosides is discussed.     

The Cold Origin of Life: B. Implications Based on Pyrimidines and Purines Produced From Frozen Ammonium Cyanide Solutions

A wide variety of pyrimidines and purineswere identified as products of a dilute frozen ammoniumcyanide solution that had been held at –78°C for 27 years.This demonstrates that both pyrimidines and purines couldhave been produced on the primitive earth in a short time byeutectic concentration of HCN, even though the concentrationof HCN in the primitive ocean may have been low. We suggestthat eutectic freezing is the most plausible demonstratedmechanism by which HCN polymerizations could have producedbiologically important prebiotic compounds.     

Improved nitrogen removal by application of new nitrogen-cycle bacteria

In order to meet increasingly stringentEuropean discharge standards, new applicationsand control strategies for the sustainableremoval of ammonia from wastewater have to beimplemented. In this paper we discuss anitrogen removal system based on the processesof partial nitrification and anoxic ammoniaoxidation (anammox). The anammox process offersgreat opportunities to remove ammonia in fullyautotrophic systems with biomass retention. Noorganic carbon is needed in such nitrogenremoval system, since ammonia is used aselectron donor for nitrite reduction. Thenitrite can be produced from ammonia inoxygen-limited biofilm systems or in continuousprocesses without biomass retention. Forsuccessful implementation of the combinedprocesses, accurate biosensors for measuringammonia and nitrite concentrations, insight inthe complex microbial communities involved, andnew control strategies have to be developed andevaluated.     

Degraded Softwater Lakes: Possibilities for Restoration

In the Netherlands, the characteristic flora of shallow softwater lakes has declined rapidly as a consequence of eutrophication, alkalization and acidification. The sediment of most lakes has become nutrient rich and anaerobic. We expected that, if a vital seed bank was still present, restoration of the original water quality and sediment conditions would lead to the return of softwater macrophytes. The restoration of 15 degraded, shallow, softwater lakes in the Netherlands was monitored from 1983 to 1998. In eutrophied as well as in acidified lakes, removal of accumulated organic matter from the sediment and shores was followed by rapid recolonization of softwater macrophytes present in the seedbank. After isolation from alkaline water and subsequent mud removal, this recovery was also observed in alkalized lakes. Further development of softwater vegetation correlated strongly with the water quality. When renewed eutrophication was successfully prevented, softwater macrophytes could expand. However, in acidified lakes, Juncus bulbosus and Sphagnum species became dominant after restoration. Liming of an acidified lake was followed by re‐acidification within 3 years. Recolonization by softwater macrophytes was inhibited by high turbidity of the water column and spreading of large helophytes on the shore. As an alternative, controlled inlet of alkaline, nutrient‐poor groundwater was studied in a few lakes. The pH of those lakes increased, the carbon and nitrogen availability decreased and softwater macrophytes returned. Successful restoration has contributed considerably to maintaining biodiversity in softwater lakes in the Netherlands.