Polysaccharide concentration and molecular weight effects on the oxygen mass transfer in a reciprocating plate bioreactor

In most polysaccharide fermentations, the nature of the fermentation broth changes drastically with time and, as a result, the overall oxygen mass transfer coefficient (K(L)a) can vary by orders of magnitude. To obtain a better understanding of this phenomenon, an experimental program was devised to study the respective influence of molecular weight and concentration of dextran solutions on K(L)a. Experiments were conducted in a reciprocating plate bioreactor. This bioreactor uses a stack of perforated plates that is reciprocated axially in the column and it is therefore well suited for mixing viscous liquid broths and providing uniform overall mass transfer coefficients. The variation of K(L)a with the power input per unit volume and the superficial gas velocity were obtained for three ranges of molecular weights and five concentrations of dextran. In every medium, two regimes of operation were observed as a function of the power input per unit volume: a first regime, at low power inputs per unit volume where K(L)a remains constant until a threshold of power input is attained; and a second regime, which is characterized by a steep increase of K(L)a as a function of the power input per unit volume. The presence of dissolved biological macromolecules, not only because of their effect on the rheology of the medium but also because their effect on the gas-liquid interface, has a significant impact on K(L)a. It was found that, generally, small concentrations of polysaccharide favor oxygen mass transfer despite the increase in medium viscosity. However, the respective influence of polysaccharide concentration and molecular weight was different for the two regimes of operation. (c) 1996 John Wiley & Sons, Inc.     

Assay of the antiangiogenic compound TNP-470, and one of its metabolites, AGM-1883, by reversed-phase high-performance liquid chromatography in plasma

This paper describes a reversed-phase, high-performance liquid chromatographic (HPLC) method for the isolation, detection, and quantification of TNP-470 (I) and one of its active metabolites, AGM-1883 (II), from plasma. These compounds are initially extracted from plasma with an organic solvent and then separated from one another on a C₁₈ column. Those fractions eluting from the C₁₈ column and containing either I or II are then derivatized through their epoxide moieties with sodium 8-quinolinethiolate (SQT). This derivatization produces fluorescent species that are isolated and quantified by a second reversed-phase HPLC analysis. The assay yields a lower limit of reliable quantification of 2.5 ng/ml and is linear to a concentration at least as high as 160 ng/ml. The inter-assay percent coefficient of variation is less than 18%.     

The Total Chemical Synthesis of Monocyte Chemotactic Protein-1 (MCP-1)

The affinity-based N^α-amino protecting group tetrabenzo [a,c,g,i]fluorenyl-17-methoxycarbonyl (Tbfmoc) has been utilized as a hydrophobic probe to allow the simple, quick and highly effective isolation of a 76 residue cysteine-containing protein (MCP-1). The base-labile Tbfmoc group can be removed under very mild conditions, which preserve the thiol-con taining protein in the reduced state. Oxidative folding was then used to furnish the biologically active β-chemokine MCP-1.     

Glucose Transporter (GLUT-4) Is Targeted to Secretory Granules in Rat Atrial Cardiomyocytes

The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the transGolgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulindependent movement to the cell surface. To examine the relationship between these GLUT-4–containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed.     

Radioimmunoassay of atrial natriuretic factor (ANF) in rat atria

We describe a solid phase radioimmunoassay for atrial natriuretic factor (ANF) and its application for measurement of this peptide in homogenates of rat atria. The method uses a synthetic 26 amino-acid fragment (8-33 ANF) of the native peptide. Sample (or standard) are incubated with the rabbit anti-8-33 ANF antiserum in peptide (8-33 ANF)-coated wells. Then an excess of I125 goat anti-rabbit IgG is added. The radioactivity bound is directly proportional to the amount of ANF present. The concentration of immunoreactive ANF has been found to be about 4 times higher in the right atrium than in the left atrium of the rat.     

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.     

IN VITRO ALLELOPATHIC INHIBITION OF NITRIFICATION BY BALSAM POPLAR AND BALSAM FIR

A soil percolation apparatus was used to study the effect of plant extracts on nitrification. Foliar leachates, leaf extracts, and bud extracts of balsam fir (Abies balsamea) and balsam poplar (Populus balsamifera) were added to the ammonium percolation solution. Color tests and quantitative measurement of residual ammonium and formed nitrate were applied. Foliar leachates of balsam fir strongly inhibited nitrification; leachates of balsam poplar were slightly less inhibitory. Balsam fir needle and balsam poplar dormant bud extracts (5% w/v) completely prevented the oxidation of ammonium. The same estracts (2% w/v) still prevented the oxidation of ammonium to nitrate to a large extent. Once again balsam fir extracts inhibited the process more strongly than balsam poplar. No evidence was obtained for the presence of a specific inhibitor of nitrifier microorganisms.     

Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum. Complete sequence and localization of the active-site serine.

A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families.     

Interleukin 2 production in iron-deficient children

The relationship between iron status and capacity for IL-2 production by lymphocytes was assessed in 81 children from 6 mo to 3 yr of age selected at random from a population with low socioeconomic status, undergoing free systematic examination in four children's health centers in the Paris area. Iron deficiency was defined by the existence of at least two abnormal values among the three indicators of iron status: serum ferritin level ≤12 μg/L, transferrin saturation <12%, and erythrocyte protoporphyrin concentration >3 μg/g hemoglobin. According to this definition, 53 children were classified as iron deficient and 28 as iron sufficient. No differences were observed between the iron-deficient and iron-sufficient groups in terms of the IL-2 concentration without stimulation by PHA. IL-2 production by lymphocytes stimulated with PHA, as well as the stimulation index (ratio of IL-2 concentration following stimulation by PHA to that of IL-2 concentration without stimulation by PHA) were significantly lower in iron-deficient children. The reduction in IL-2 production by activated lymphocytes observed in our study of iron-deficient children may be responsible for impairments in immunity found by other authors, particularly in cell-mediated immunity.