Proteins of the extracellular fluid of mouse brain: extraction and partial characterization

An extraction procedure for the isolation of proteins from the brain extracellular fluid (ECF) was developed and applied to studies of the ECF components of mouse brain. Perfused intact brains were incubated in an isotonic medium for periods of up to 2 h at 0 degrees C to allow the release of ECF into the medium without disruption of the integrity of the tissue. The validity of the extraction procedure was established by (a) the fact that the total yield of ECF proteins was constant per unit weight of brain tissue, (b) the absence of tyrosine hydroxylase, an enzyme marker of the cytoplasmic fraction, from the extracts, and (c) the distinctive features of the one- and two-dimensional gel electrophoretic patterns of ECF proteins as compared with those of the cytoplasmic and membrane fractions. The results indicate that the extracellular fluid of mouse brain contains a mixture of proteins with a wide distribution of molecular weights (10,000-100,000 daltons) at a concentration level of about 0.3%.     

Formation of 13,14-dihydro-prostaglandin E1 during intravenous infusions of prostaglandin E1 in patients with peripheral arterial occlusive disease.

Formation of 13,14-dihydro-prostaglandin (PG) E1 during intravenous infusions of PGE1 in patients with peripheral arterial occlusive disease was investigated. Using both high performance liquid chromatography (h.p.l.c.) combined with radioimmunoassay and gas chromatography/triple stage quadrupole mass spectrometry (GC/MS/MS) basal levels of 13,14-dihydro-PGE1 were found to be close to or below the detection limits of the assay methods. Levels of the PGE1 metabolite increased significantly during the infusion periods and decreased after their end. Since 13,14-dihydro-PGE1, in contrast to its precursors 15-keto-PGE1 and 15-keto-13,14-dihydro-PGE1, is biologically active, its formation could contribute to the beneficial effects of PGE1 administered intravenously in patients with peripheral arterial occlusive disease.     

The effect of graded renal nerve stimulation on renal function in the anaesthetized rabbit

The renal nerves of the rabbit were stimulated to cause less than 5, 9 or 22% reduction in renal blood flow. Glomerular filtration rate was reduced by 0, 3 and 11% respectively when the renal nerves were stimulated at these increasing rates. At low and moderate rates of renal nerve stimulation absolute and fractional sodium excretions were reduced by between 17 and 22%, but at the highest rates they were reduced by 55 and 53% respectively. At increasing rates of renal nerve stimulation plasma renin activity was increased by 40, 70 and 180%.     

Actinomycetes scale‐up for the production of the antibacterial,nocathiacin

An Amycolatopsis fastidiosa culture, which produces the nocathiacin class of antibacterial compounds, was scaled up to the 15,000 L working volume. Lower volume pilot fermentations (600, 900, and 1,500 L scale) were conducted to determine process feasibility at the 15,000 L scale. The effects of inoculum volume, impeller tip speed, volumetric gas flow rate, superficial gas velocity, backpressure, and sterilization heat stress were examined to determine optimal scale‐up operating conditions. Inoculum volume (6 vs. 2 vol %) and medium sterilization (R_o of 68 vs. 92 min^?1) had no effect on productivity or titer, and higher impeller tip speeds (2.1 vs. 2.9 m/s) had a slight effect (20% decrease). In contrast, higher backpressure, incorporating increased head pressure at the 15,000 L scale (1.2 vs. 0.7 kg/cm²) and low gas flow rates (0.25 vs. 0.8 vvm), appeared to be problematic (40–50% decrease). High off‐gas CO₂ levels were likely reasons for observed lower productivity. Consequently, air flow rate for this 25‐fold scale‐up (600–15,000 L) was controlled to match off‐gas CO₂ profiles of acceptable smaller scale batches to maintain levels below 0.5%. The 15,000 L‐scale fermentation achieved an expected nocathiacin I titer of 310 mg/L after 7 days. Other on‐line data (i.e., pH, oxygen uptake rate, and CO₂ evolution rate) and off‐line data (i.e., analog production, glucose utilization, ammonium production, and dry cell weight) at the 15,000 L scale also tracked similarly to the smaller scale, demonstrating successful fermentation scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Pattern morphogenesis in cell walls of diatoms and pollen grains: a comparison

Summary Mechanisms acting in pattern morphogenesis in the cell walls of two distant groups of plants, pollen of spermatophytes and diatoms, are compared in order to discriminate common principles from plant group- and wall material-specific features. The exinous wall in pollen is sequentially deposited on the exocellular side of the plasmalemma, while the siliceous wall in diatoms is formed intracellularly within an expanding silica deposition vesicle (SDV) which is attached to the internal face of the plasmalemma. Two levels of patterning occur in diatom and pollen walls: the overall pattern stabilises the wall mechanically and is apparently initiated in both groups by the parent cell, and a microtubule-dependent aperture and portula pattern created by the new mitotic (diatoms) or meiotic (pollen) cells. The parent wall in diatoms, and also the callosic wall in microspores, functions as anchor surfaces for transient, species-specific patterned adhesions of the plasmalemma to these walls, involved in pattern and shape creation. Patterned adhesion and exocytosis is blocked in pollen walls where the plasmalemma is shielded by the endoplasmic reticulum at the sites of the future apertures. In diatoms, wall patterning is uncoupled from the formation of a siliceous wall per se when the SDV and its wall is formed without contact to the the plasmalemma. Conversely, a blue-print pattern laid out in advance along the plasmalemma can be found in several diatoms. This highlights the key function of the plasmalemma and its associated membrane skeleton (fibrous lamina), and its orchestrated co-operation with elements of the radial filamentous cytoskeleton (actin?) in pattern formation. The role of microtubules during generation of the overall pattern may be primarily a transport and stabilizing function. Auxiliary organelles (spacer vesicles, endoplasmic reticulum, mitochondria) involved in diatoms for shaping the SDV, and a mechanism adhering and disconnecting this SDV together with spacer organelles in a species-specifically controlled sequence to and from the plasmalemma, are unnecessary for pollen wall patterning. The precise positioning of the portula pattern in diatom walls is discussed with respect to their role as permanent anchors of the cytoplasm to its wall, and in providing spatial information for nucelar migration and the next cell division, whereas apertures in pollen are for single use only.Abbreviations AF actin filaments - C/Ca callose - CF cleavage furrow - cPL cleavage plasmalemma - DV dense vesicles - ER endoplasmic reticulum - ET epitheca - HT hypotheca - mPL folded plasmalemma - MT microtubules - MTOC microtubule organising centre - PEV primexine (matrix) vesicles - PL plasmalemma - SDV silica deposition vesicle - Si silica - SL SDV-membrane - SPV spacer vesicles Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Effect of glucose on assimilatory sulphate reduction in Arabidopsis thaliana roots

With the aim of analysing the relative importance of sugar supply and nitrogen nutrition for the regulation of sulphate assimilation, the regulation of adenosine 5'-phosphosulphate reductase (APR), a key enzyme of sulphate reduction in plants, was studied. Glucose feeding experiments with Arabidopsis thaliana cultivated with and without a nitrogen source were performed. After a 38 h dark period, APR mRNA, protein, and enzymatic activity levels decreased dramatically in roots. The addition of 0.5% (w/v) glucose to the culture medium resulted in an increase of APR levels in roots (mRNA, protein and activity), comparable to those of plants kept under normal light conditions. Treatment of roots with d-sorbitol or d-mannitol did not increase APR activity, indicating that osmotic stress was not involved in APR regulation. The addition of O-acetyl-l-serine (OAS) also quickly and transiently increased APR levels (mRNA, protein, and activity). Feeding plants with a combination of glucose and OAS resulted in a more than additive induction of APR activity. Contrary to nitrate reductase, APR was also increased by glucose in N-deficient plants, indicating that this effect was independent of nitrate assimilation. [35S]-sulphate feeding experiments showed that the addition of glucose to dark-treated roots resulted in an increased incorporation of [35S] into thiols and proteins, which corresponded to the increased levels of APR activity. Under N-deficient conditions, glucose also increased thiol labelling, but did not increase the incorporation of label into proteins. These results demonstrate that (i) exogenously supplied glucose can replace the function of photoassimilates in roots; (ii) APR is subject to co-ordinated metabolic control by carbon metabolism; (iii) positive sugar signalling overrides negative signalling from nitrate assimilation in APR regulation. Furthermore, signals originating from nitrogen and carbon metabolism regulate APR synergistically.     

A micro-scale process for high-throughput expression of cDNAs in the yeast Saccharomyces cerevisiae

Methods have been developed aimed at applying at high-throughput technology for expression of cloned cDNAs in yeast. Yeast is a eukaryotic host, which produces soluble recombinant proteins and is capable of introducing post-translational modifications of protein. It is, thus, an appropriate expression system both for the routine expression of various cDNAs or protein domains and for the expression of proteins, which are not correctly expressed in Escherichia coli. Here, we describe a standard system in Saccharomyces cerevisiae, based on a vector for intracellular protein expression, where the gene products are fused to specific peptide sequences (tags). These epitope tags, the N-terminal His(6) tag and the C-terminal StrepII tag, allow subsequent immunological identification and purification of the gene products by a two-step affinity chromatography. This method of dual-tagged recombinant protein purification eliminates contamination by degraded protein products. A miniaturization of the procedures for cloning, expression, and detection was performed to allow all steps to be carried out in 96-well microtiter plates. The system is, thus, suitable for automation. We were able to analyze the simultaneous protein expression of a large number of cDNA clones due to the highly parallel approach of protein production and purification. The microtiter plate technology format was extended to quantitative analysis. An ELISA-based assay was developed that detects StrepII-tagged proteins. The application of this high-throughput expression system for protein production will be a useful tool for functional and structural analyses of novel genes, identified by the Human Genome Project and other large-scale sequencing projects.