Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.     

Binding of anti-human-interleukin-6 monoclonal antibodies to synthetic peptides of human interleukin-6 studied using surface plasmon resonance.

Biosensor technology employing surface plasmon resonance (SPR) detection provides a highly-sensitive (sub ng), non-extrinsic labelling approach for monitoring protein interactions in real-time. We have used this approach to map the binding sites on human interleukin-6 (hIL-6) for a series of anti-hIL-6 monoclonal antibodies (mAbs). Epitopes were localised by monitoring the ability of ten synthetic peptides, spanning the sequence of hIL-6, to inhibit the binding of anti-hIL-6 mAbs to immobilised hIL-6. Peptide P8 (Pro139-Gln153) inhibited binding of anti-IL-6-mAbs 1, 2 and 7. To increase the sensitivity of detection of antibody-synthetic peptide interactions, a procedure was developed for immobilising the synthetic peptides directly to the sensor surface of the SPR instrument. From this study, association equilibrium constants of 2.1 x 10(6)M-1 and 3.6 x 10(4)M-1 were calculated for the mAb7-immobilised P8 and mAb7-free P8 interactions, respectively.     

Kringle domains and plasmin denaturation.

The rate of plasmin denaturation was in the order of Lys-plasmin greater than miniplasmin greater than microplasmin. Fibrinogen degradation products (FDP) dose dependently increased the denaturation rate of Lys-plasmin and mini-plasmin with a maximal rate constant at the FDP/plasmin ratio of about 0.5. The denaturation rate constant of microplasmin was not affected. FDP increased the rate of plasmin denaturation was in parallel with its effect on the interaction among kringle domains. Without FDP only trace amounts of plasminogen dimer could be detected by cross-linking with bis-(sulfo-succinimidyl)-suberate followed by SDS gel electrophoresis. In the low concentration of FDP significant amounts of oligomers of Glu-, mini-plasminogens, kringle 1-3 and kringle 1-5 were observed. High concentration of FDP, however, decreased plasminogen oligomer.     

In vivo mechanical properties of leukocytes during adhesion to venular endothelium

Transient deformations of leukocytes (WBCs) were studied during their saltation along post-capillary venous endothelium (EC) in mesentery of the rat. During intermittent adhesion of WBCs to EC, prevailing fluid shear stresses, tau wall, resulted in a stepwise loading of the WBC upon attachment with a transient increase in length, L(t), and reduction in height, H(t). Measurements of L(t) and H(t) from frame-by-frame analysis of video recordings were modelled as the simple shear of a standard linear viscoelastic solid to facilitate calculation of the elastic (k1, k2) and viscous (mu) elements with k1 in parallel with serial elements k2 and mu. The magnitude of tau wall was determined from measurements of red cell velocity within the venule. During the spontaneous adhesion of WBCs, a value of cell viscosity (mu) of 45 Poise was determined. Stimulating adhesion by topical application of the chemoattractant FMLP resulted in a 15-fold increase of mu to 668 Poise. Transient deformations during topical application of cytochalesin B to disrupt actin fibers within the WBC, yielded a 40% reduction in k1, compared to an 80% reduction with colchicine which disrupts the microtubule structure. Thus, colchicine treated cells appear to be twice as deformable as cells treated with cytochalesin. During adhesion stimulated by the cytokine Interleukin-1, mu increased 50% without changes in k1 and k2, possibly due to slight activation of the WBC.     

人αA干扰素在克鲁氏乳酸酵母中的表达和分泌

pE1是由酵母天然质粒pKD1衍生出来的重组穿梭质粒,在克鲁氏乳酸酵母中具有高拷贝,高稳定性等特点。把人αA干扰素分泌表达单元克隆到pE1载体中,得到分泌型表达质粒pE-IFN1。pE-IFN1在克鲁氏乳酸酵母中相当稳定,在非选择性培养基中生长50世代后,大多数酵母细胞仍带有质粒。结果表明,分泌表达单元中的酿酒酵母α因子的分泌信号肽能被克鲁氏乳酸酵母的蛋白质分泌系统所识别,人αA干扰素被分泌到细胞外。在摇瓶培养条件下每升发酵液中含有1—2毫克αA干扰素。     

Interaction of lipoproteins with isolated ovary plasma membranes

Plasma membranes of ovarian luteal and adrenal cortical cells from "microvillar channels," a unique extracellular compartment formed by the close apposition of flattened microvillar surfaces. Microvillar channels have unusual affinity for cholesterol-rich lipoproteins, and, in vivo, may provide an increased surface area for these particles. In this research, we have isolated a plasma membrane-enriched fraction from rat luteinized ovaries, in which closely apposed membrane (i.e. microvillar channels) comprise about 30% of the preparation. Following in vitro incubations (approximately 1 h) of this plasma membrane fraction with different plasma lipoproteins, the closely apposed plasma membrane surfaces widen and become filled with lipoprotein particles (up to about 30 nm), whereas other membranes of the fraction show little binding. Competition experiments show that rat high density lipoproteins have the highest affinity for binding to the plasma membrane fraction. Radiolabeled plasma lipoprotein and the tissue-specific hormone, human chorionic gonadotropin, showed specific and saturable binding to the plasma membrane fraction, whereas other macromolecules used as controls did not. Radioautographic analyses of 125I-labeled lipoproteins and human chorionic gonadotropin indicate that binding occurs predominantly to the closely apposed plasma membranes (i.e. microvillar channels of the fraction). These studies show that microvillar channels of steroid-secreting cells entrap large numbers of plasma lipoproteins, particularly high density lipoproteins particles, presumably functioning in the delivery of cholesterol to these cells.     

Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine

Replacement of the arginine-138 of adenylate kinase (AK) by lysine or methionine resulted in a decrease in kcat by a factor of 10(4), increases in Km by a factor of 10-20, and relatively little changes in dissociation constants. Proton nuclear magnetic resonance (NMR) studies were then undertaken to obtain structural information for quantitative interpretation of the kinetic data. Since the lysine mutant (R138K) represents a conservative mutation with surprisingly large effects on kinetics, structural studies were focused on the wild type (WT) and R138K. The results and conclusions are summarized as follows: (i) The aromatic spin systems of WT and R138K were assigned from total correlated spectroscopy (TOCSY). Comparison of the chemical shifts of aromatic protons, one-dimensional spectra, TOCSY, and nuclear Overhauser enhanced spectroscopy (NOESY) indicated that the conformation of R138K was almost unperturbed relative to that of WT. Thus Arg-138 is not important for the tertiary structure. (ii) Proton NMR titrations with AMP and MgATP suggested that substrate binding affinities and substrate-induced conformational changes are nearly identical between WT and R138K. Thus arginine-138 should not be involved in stabilizing the first substrate in the binary complex. (iii) Notable differences were observed between the proton NMR spectra of the WT and R138K complexes with the reaction mixture, which agrees with the perturbation in the Km values of R138K. The differences were analyzed in detail by using a "static reaction mixture'--p1, p5-bis(5'-adenosyl)pentaphosphate (MgAP5A). The aromatic spin systems of WT + MgAP5A and R138K + MgAP5A were partially assigned from various two-dimensional spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exogenous tenascin inhibits mesodermal cell migration during amphibian gastrulation.

We have used amphibian gastrulation as a model system to study the action of the extracellular matrix (ECM) glycoprotein tenascin on mesodermal cell migration. Tenascin function was assayed in vitro during spreading of isolated cells from the dorsal marginal zone (DMZ) and during cell migration from DMZ explants. Plastic coated with bovine fibronectin or gastrula ECM was used as a substratum. In both cases, tenascin added to the medium inhibited spreading and migration of mesodermal cells. In addition, a substratum coated with a mixture of fibronectin and tenascin was found to prevent mesodermal cell migration. Tenascin was also microinjected into the blastocoel cavity of living embryos at the late blastula stage. This led to a complete arrest of gastrulation in more than 80% of the cases. Scanning electron microscopy of fractures from arrested gastrulae showed that mesodermal cell migration was blocked. Similar injection experiments carried out at the middle gastrula stage demonstrated that tenascin is able to inhibit cell migration after cells have already contacted the ECM. Mesodermal cell migration in the presence of tenascin could be restored in vitro and in vivo by the monoclonal antibody mAb Tn68 which is known to mask a cell binding site of the molecule. Finally, tenascin microinjected into the blastocoel of blastula or gastrula stage embryos bound within 15 min to the ECM fibrils at all the stages studied. Our results show that exogenous tenascin can be incorporated into embryonic ECM and interferes in vivo with the interactions of cells with a fibronectin-rich matrix.