C nuclear magnetic resonance study of acetate incorporation into malate during ca-uptake by isolated leaf tissues

¹³C Nuclear magnetic resonance spectroscopy of leaflets of Gleditsia triacanthos and Albizia julibrisin was used to determine the fate of acetate taken up during the absorption of calcium from ¹³C-labeled Ca-acetate solution. Small amounts of acetate accumulated temporarily in the leaf tissues, but the bulk of acetate was incorporated into malate. The initial rate of malate synthesis was very low, but increased rapidly during acetate treatment and reached its maximum after 8 hours; the enzymes involved in malate synthesis thus appear to be substrate induced. Use of acetate-2-¹³C yielded malate labeled in C-3, indicating that vacuolar malate accumulating during Ca-uptake might be synthesized via malate synthase from acetate and glyoxalate. However, a source of glyoxalate condensing with acetate during malate synthesis could not be identified. Glycolate produced in photorespiration is an unlikely source, because glycolate-2-¹³C was absorbed and metabolized by the leaf tissues into products of the glycolate pathway, but was not a major precursor in malate synthesis. Malate synthesis via the glyoxalate cycle is also unlikely, because no evidence for the recycling of a ¹³C-labeled 4-carbon organic acid was found. Malate synthesis in the leaflets of Gleditsia and Albizia thus appears to involve the inducible condensation of acetate with a 2-carbon compound of unidentified nature and origin.     

Improved biocatalyst effectiveness by controlled immobilization of enzymes

Nonuniform enzyme distributions can be obtained by kinetic control of the immobilization process. Such heterogeneous biocatalysts exhibit higher effectiveness compared to conventional immobilization procedures, when the mass transfer of substrates or products is limiting. Model calculations provide some insight into the relative weight of the immobilization parameters with respect to optimal control of the enzyme distribution. Experimental results and model calculations show that considerably improved effectiveness of biocatalysts can be obtained. The role of external mass transfer is emphasized.     

Establishment of microscale vegetation pattern in maritime chaparral after fire

We quantified microscale pattern in vegetation and seed assemblages along a 24 m transect before and for two years following a controlled burn in chamise (Adenostoma fasciculatum) chaparral in central coastal California. Our objective was to document scale-dependent correlation between pre-burn seed assemblages, pre-burn canopy cover, microtopography, soil temperatures during burning, post-burn seed assemblages and post-burn vegetation. Scale-dependent correlations among pre- and post-burn seed densities, maximum soil temperatures during burning, microtopography and post-burn vegetation were measured based on two-term local covariance analysis.Seed distribution varied among species prior to fire, with seeds of some annual species concentrated in gap areas. Maximum soil temperatures during burning ranged from less than 50 °C to 225 °C, and were generally lowest in gaps in the pre-burn canopy. These gaps were associated with local topographic depressions. After burning, readily germinable seeds were concentrated in or near gaps in the pre-burn canopy. Germination of different species was variously enhanced, diminished or unchanged by the passage of fire. Post-burn vegetation was very patchy, with some areas nearly devoid of seedlings and other areas, especially pre-burn canopy gaps, supporting numerous seedlings. Seedling recruitment patterns in the second year were generally highly correlated with patterns in pre-burn seed banks and first year vegetation. Although many species exhibited similar recruitment patterns, several different mechanisms may have been responsible for the origin of those patterns.     

Long lasting anti-adrenergic effect of 7-oxo-prostacyclin in the heart: a cycloheximide sensitive increase of phosphodiesterase isoform I and IV activities

Evidence is accumulating that 7-oxo-prostacyclin (7-oxo-PGI₂) induces a delayed indirect anti-adrenergic and cytoprotective effect on the myocardium, the mechanism of which is still unclear. To demonstrate that a single application of 7-oxo-PGI₂ (50 g/kg i.m.) 48 h prior to starting experiments attenuates the isoprenaline inducible inotropic response and accumulation of cAMP, isolated hearts of pretreated animals were perfused in the Langendorff mode with and without isoprenaline (1 to 100 nM). The late anti-adrenergic effect of the drug was manifested by a significant attenuation in the elevation of cAMP levels as well as in contractile force development. This effect was not due to changes in cAMP generation as there were identical ₁-adrenoceptor densities and affinities (as calculated from [³H]-CGP binding studies), G_i and G_s protein patterns (as taken from Western blots) as well as adenylyl cyclase activity measurements in the hearts studied. The anti-adrenergic potency of 7-oxo-PGI₂, however, was found to be related to a significant rise in cyclic nucleotide hydrolysis by phosphodiesterase (PDE). Using the fast-performance liquid chromatographic separation for PDE isoforms, a significant increase in the activity of PDE isoforms I and IV (260±28 vs 110±12 pmol cGMP/min x enzyme fraction and 77±11 vs 34±3 pmol cAMP/min x enzyme fraction, respectively) was found in the solubilized fraction of cardiac membranes in comparison to untreated controls; PDE IV activity was also increased in the cytosolic fraction (106±14 vs 65±6 pmol cAMP/min x enzyme fraction). The hypothesis that the delayed anti-adrenergic effect of 7-oxo-PGI₂ is initiated by an induction and accelerated synthesis of PDE I and IV in the heart is underlined by the fact that cycloheximide suppresses completely both the rise in PDE activities and the anti-adrenergic effects studied. It is suggested that an inducible predominance of cAMP degradation over its generation may be of relevance in processes related to heart protection.     

ADP/ATP Translocator from Pea Root Plastids (Comparison with Translocators from Spinach Chloroplasts and Pea Leaf Mitochondria)

The kinetic properties of the adenosine 5[prime]-diphosphate/adenosine 5[prime]-triphosphate (ADP/ATP) translocator from pea (Pisum sativum L.) root plastids were determined by silicone oil filtering centrifugation and compared with those of spinach (Spinacia oleracea L.) chloroplasts and pea leaf mitochondria. In addition, the ADP/ATP transporting activities from the above organelles were reconstituted into liposomes. The Km(ATP) value of the pea root ADP/ATP translocator was 10 [mu]M and that for ADP was 46 [mu]M. Corresponding values of the spinach ADP/ATP translocator were 25 [mu]M and 28 [mu]M, respectively. Comparable results were obtained for the reconstituted ATP transport activities. The transport was highly specific for ATP and ADP. Adenosine 5[prime]-monophosphate (AMP) caused only a slight inhibition and phosphoenolpyruvate and inorganic pyrophosphate caused no inhibition of ATP uptake. With pea root plastids and spinach chloroplasts, Km values >1 mM were obtained for ADP-glucose. Since the concentrations of ATP and ADP-glucose in the cytosolic compartment of spinach leaves have been determined as 2.5 and 0.6 mM, respectively, a transport of ADP-glucose by the ADP/ATP translocator does not appear to have any physiological significance in vivo. Although both the plastidial and the mitochondrial ADP/ATP translocators were inhibited to some extent by carboxyatractyloside, no immunological cross-reactivity was detected between the plastidial and the mitochondrial proteins. It seems probable that these proteins derive from different ancestors.     

Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.     

Recent domoic acid closures of shellfish harvest areas in Washington State inland waterways

Several species of the toxigenic diatom Pseudo-nitzschia, together with low concentrations of domoic acid (DA) in shellfish have been observed in Puget Sound, Washington State, since 1991. However, for the first time in September 2003, high-density blooms of Pseudo-nitzschia forced the closure of recreational, commercial, and tribal subsistence shellfish harvesting in Puget Sound. Here we report on the environmental conditions associated with shellfish closures in two inland waterways of Washington State during the Fall 2005. In Sequim Bay, shellfish harvest losses occurred on September 12 following the measurement of elevated macronutrient levels on September 2, and a bloom of P. pseudodelicatissima (up to 13 million cells/L) on September 9. Ambient NH₄ concentrations >12 μM (measured on September 2) were likely due to anthropogenic sources, ostensibly from sewage inputs to Sequim Bay. The closure of a Penn Cove commercial shellfish farm on October 16 was caused by a bloom of P. australis that followed a period of sustained precipitation, elevated Skagit River flow, and persistent southeasterly winds. The relative importance of a number of environmental factors, including temperature, stratification caused by rivers, and nutrient inputs, whether natural or anthropogenic, must be carefully studied in order to better understand the recent appearance of massive blooms of toxigenic Pseudo-nitzschia in the inland waterways of Washington State.