Nutrient-split feeding strategy for dialysis cultivation of Escherichia coli

Reduction in nutrient loss during dialysis cultivation of Escherichia coli on a glycerol medium was investigated. A dialysis reactor with an inner fermentation and an outer dialysis chamber was used. Aerobic condition was maintained by limiting the glycerol feed rate to an optimum value which was estimated from the oxygen requirements for glycerol oxidation and oxygen transfer capacity of the reactor. High reduction in nutrient loss was achieved by using water as the dialyzing fluid. However, osmotic movement of water from the dialysis to the fermentation chamber was observed, and the final cell concentration was low. With a nutrient-split feeding strategy (feeding glycerol directly to the fermentation chamber and dialyzing with salt solution), glycerol loss was small, there was no osmotic flux of water to the fermentation chamber, and the cell concentration was high. Both glycerol and salt loss could be avoided, and a cell concentration of 170 g/L was obtained when the dialysis process was substituted by addition of XAD adsorbents to the dialysis chamber. Application of this nutrient-split feeding strategy to cell cultivation in a stirred tank reactor, coupled with dialysis in external dialyzer modules, resulted in low cell concentrations. (c) 1993 Wiley & Sons, Inc.     

Comparison of alternative extraction methods for secretome profiling in human hepatocellular carcinoma cells

Secreted proteins are important sources for early detection and diagnosis of disease, and as such have received considerable attention. The extraction of low concentration proteins from large volumes of culture media, which are rich in salts and other compounds that interfere with most proteomics techniques, presents a problem for secretome studies. Ultrafiltration, precipitation, and dialysis are three major extraction methods that can be used to overcome this problem. The present study for the first time, compared the merits and shortcomings of these three methods, without bias. Centrifugal ultrafiltration provided the best extraction efficiency, and precipitation provided the highest number of identifiable proteins. The three methods yielded closely related, but different, information on the secretome; thus, they should be considered complementary or, at least, supplementary methods. Three hundred and sixty unique proteins were identified, including 211 potential secreted proteins. Compared with previous studies, this study also identified 42 new secreted proteins. The present study not only offers a reference for the selection of secretome extraction methods, but also expands the secretome database for the investigation of hepatocellular carcinoma.     

Extracellular Overflow of Neuroactive Amino Acids During Severe Insulin-Induced Hypoglycemia: In Vivo Dialysis of the Rat Hippocampus

Hypoglycemia-evoked changes in levels of extracellular excitatory and inhibitory amino acids were studied using the microdialysis technique. A newly designed dialysis probe was inserted stereotaxically into the rat hippocampus. Animals were then subjected to insulin-induced hypoglycemia; then blood glucose levels were restored by glucose injections after a 30-min period of isoelectric electroencephalography. Dialysates were collected before, during, and after the isoelectric period. Amino acids in the dialysates were analyzed by liquid chromatography and fluorescence detection following automatic precolumn derivatization with o-phthaldialdehyde. During the isoelectric phase, the concentration of aspartate increased 15-fold, whereas glutamate, gamma-amino-butyric acid, taurine, and phosphoethanolamine levels were elevated three- to sixfold. Smaller increases were observed for nonneuroactive amino acids such as asparagine, alanine, and phenylalanine. In contrast to all other amino acids, the glutamine content was reduced to less than 30% of preisoelectric values. The concentrations of the neuroactive amino acids were restored to normal in the post-isoelectric phase. These data demonstrate that there is an extracellular overflow of neuroactive amino acids, especially aspartate, during severe hypoglycemia.     

Comparison of serum free thyroxine measurements by chemiluminescence and equilibrium dialysis

A study of 239 patients compared free thyroxine (FT4) measurements made by equilibrium dialysis (ED) with measurements made using the Magic Lite FT4 chemiluminescence (Cl) immunoassay (Ciba Corning Immunodiagnostics). Patient groups: 41 normals; 27 hyperthyroid; 29 hypothyroid; 37 sick euthyroid; 10 chronic renal failure (CRF) and 25 pregnant patients; 13 oestrogen; 10 heparin; 12 salicylate; and 9 dilantin-treated patients; 3 lipaemic; 5 haemolysed; 6 hyperbilirubinaemic patients; 6 low thyroid binding protein (TBP) and 6 high TBP level patients. The two assays gave comparable results in most groups. Both assays tended to give elevated values in heparinized patients but FT4–ED results were more obviously affected. Pregnant patients and women on oral oestrogen had higher mean values with FT4–ED. In both assays the sick euthyroid and CRF patients had mean FT4 values similar to healthy euthyroid patients; the range of values in sick euthyroid and CRF patients was similar in both assays but wider than in healthy euthyroid patients. A supplemental study of 81 unselected acutely ill patients using FT4–Cl alone confirmed the wider range of values to be anticipated in sick euthyroid patients.     

中医药防治腹膜纤维化的研究进展

腹膜透析是治疗终末期肾脏病较为有效的方法之一,但是腹膜长期暴露于非生理性的腹膜透析液中（低pH、高浓度葡萄糖以及高渗透压等）会致腹膜纤维化,从而导致腹膜结构和功能的丧失,这是患者放弃腹膜透析的原因之一。现代药理学实验证明,中医药对于腹膜纤维化的防治研究主要集中在腹膜间皮细胞的上皮间质转化方面,通过对细胞因子的作用,保护和／或改善腹膜间皮细胞的功能防治腹膜纤维化。     

Measurement of Endogenous Noradrenaline Release in the Rat Cerebral Cortex In Vivo by Transcortical Dialysis: Effects of Drugs Affecting Noradrenergic Transmission

The release of endogenous noradrenaline was measured in the cerebral cortex of the halothane-anesthetized rat by using the technique of brain dialysis coupled to a radioenzymatic assay. A thin dialysis tube was inserted transversally in the cerebral cortex (transcortical dialysis) and perfused with Ringer medium (2 microliter min-1). Under basal conditions, the cortical output of noradrenaline was stable over a period of at least 6 h and amounted to 8.7 pg/20 min (not corrected for recovery). Histological control of the perfused area revealed very little damage and normal morphology in the vicinity of the dialysis tube. Omission of calcium from the perfusion medium caused a marked drop in cortical noradrenaline output. Bilateral electrical stimulation (for 10 min) of the ascending noradrenergic pathways in the medial forebrain bundle caused a frequency-dependent increase in cortical noradrenaline output over the range 5-20 Hz. Stimulation at a higher frequency (50 Hz) resulted in a levelling off of the increase in cortical noradrenaline release. Systemic administration of the dopamine-beta-hydroxylase inhibitor bis-(4-methyl-1-homopiperazinylthiocarbonyl) disulfide (FLA 63) (25 mg/kg i.p.) markedly reduced, whereas injection of the monoamine oxidase inhibitor pargyline (75 mg/kg i.p.) resulted in a progressive increase in, cortical noradrenaline output. d-Amphetamine (2 mg/kg i.p.) provoked a sharp increase in cortical noradrenaline release (+450% over basal values within 40 min). Desmethylimipramine (10 mg/kg i.p.) produced a twofold increase of cortical noradrenaline release. Finally, idazoxan (20 mg/kg i.p.) and clonidine (0.3 mg/kg i.p.), respectively, increased and decreased the release of noradrenaline from the cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability, pKa and plasma protein binding of roscovitine

In the present investigation, the binding of roscovitine (100, 500 and 1500 ng/mL) to plasma proteins was studied at 25 and 37 degrees C by ultrafiltration and equilibrium dialysis methods. Drug stability in plasma was assessed during a 48 h at 4, 25 and 37 degrees C. The effect of thawing and freezing on drug stability was studied. The pKa of roscovitine was measured using capillary electrophoresis coupled with mass spectrometry. Roscovitine was quantified utilizing liquid chromatography and tandem mass spectrometry. Roscovitine is highly bound to plasma proteins (90%). Binding of roscovitine to human serum albumin was constant (about 90%) within concentration range studied while the binding to alpha1-acid glycoprotein decreased with increasing drug concentration indicating that albumin is more important in clinical settings. However, alpha1-acid glycoprotein might be important when plasma proteins change with disease. Protein binding was higher at 25 degrees C compared to 37 degrees C. The results obtained by equilibrium dialysis were in good agreement with those obtained by ultrafiltration. Roscovitine was stable at all temperatures studied during 48 h. Roscovitine has a pKa of 4.4 showing that the drug mainly acts like a weak mono-base. The results obtained in our studies are important prior to clinical trials and to perform pharmacokinetic studies.     

Presence of nucleoside triphosphates and calcium associated with mycobacteriophage I3

The association of nucleoside triphosphate molecules and calcium ions with purified particles of mycobacteriophage I3 has been documented. The content of nucleoside triphosphate has been determined to be 118 molecules per phage particle by equilibrium dialysis against labelled ATP or 148 molecules per phage particle by the direct determination of labelled nucleoside triphosphate. The concentration of bound Ca²⁺ exhibited a high degree of variation between different batches, which may be due to the nonspecific binding of Ca²⁺ by the virus particles. However, the tightly bound Ca²⁺ not removable by dialysis against calciumspecific chelating agent, showed a constant value of 2985 atoms/phage particle.Abbreviations EGTA Ethylene glycol-bis (-aminoethylether)-N,N¹ tetraacetic acid - PFU plaque forming unit - NTP nucleoside triphosphate     

Structure of an early native‐like intermediate of β2‐microglobulin amyloidogenesis

To investigate early intermediates of β2‐microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X‐ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co‐crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild‐type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation.