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151.
152.
Abstract Perchloroethylene (PCE) was reductively dechlorinated to trichloroethylene in a 10% anaerobic sewage sludge. About 80% of the initially added PCE (300 nmol) was dechlorinated within three weeks. The calculated rates were 250 nM and 445 nM · day−1 during the first and second weeks of incubation, respectively. The depletion of PCE varied in sludges obtained from different sources.
The role of methanogenesis in the dechlorination of PCE was evaluated by inhibiting the methanogens by addition of bromoethane sulfonic acid, a potent methanogenic inhibitor. Dechlorination of PCE was significantly inhibited in sludges amended with the inhibitor. Almost 41–48% less PCE was dechlorinated in sludges containing 5 mM BESA, indicating a relation between the two processes (methanogenesis and dechlorination). Direct proof that methanogens can transform chlorinated aliphatic compounds was obtained using axenic cultures of acetate-cleaving methanogens. Methanosarcina sp , originally isolated from a chlorophenol degrading consortium, showed significantly higher dechlorinating activity as compared to Ms. mazei . Based on these studies and other recently reported observations, it appears that methanogens/methanogenesis play an important role in the anaerobic dechlorination of chlorinated aliphatics such as PCE. 相似文献
The role of methanogenesis in the dechlorination of PCE was evaluated by inhibiting the methanogens by addition of bromoethane sulfonic acid, a potent methanogenic inhibitor. Dechlorination of PCE was significantly inhibited in sludges amended with the inhibitor. Almost 41–48% less PCE was dechlorinated in sludges containing 5 mM BESA, indicating a relation between the two processes (methanogenesis and dechlorination). Direct proof that methanogens can transform chlorinated aliphatic compounds was obtained using axenic cultures of acetate-cleaving methanogens. Methanosarcina sp , originally isolated from a chlorophenol degrading consortium, showed significantly higher dechlorinating activity as compared to Ms. mazei . Based on these studies and other recently reported observations, it appears that methanogens/methanogenesis play an important role in the anaerobic dechlorination of chlorinated aliphatics such as PCE. 相似文献
153.
Molecular genetic analysis of membrane protein topology 总被引:12,自引:0,他引:12
A transmembrane protein contains domains residing in the aqueous compartments on both sides of the membrane in which it is integrated. A determination of the topology of such a protein requires the definition of which domains lie on which side of the membrane. In E. coli, mutants and gene fusions have been used to obtain this topological information. 相似文献
154.
Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation. 相似文献
155.
The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using
continuous short circuit current (I
SC
) measurements in HCO3
−/CO2 buffered solution. Steady state I
SC
averaged 73.8 ± 3.2 μA/cm2 (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na+ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y+ and a basal amino acid transport system y+L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic
amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I
SC
averaging 36.6 ± 5.4 μA/cm2 (n= 19) and 32.0 ± 7.2 μA/cm2 (n= 8), respectively. The response was preserved in the absence of bath Cl− (n= 4), but was abolished either in the absence of apical Na+ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I
SC
response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization
of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na+ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract
passive cationic amino acid leak into the CCD in the presence of electrogenic Na+ transport, consistent with reports of stimulated expression of Na+ and cationic amino acid transporters by aldosterone.
Received: 11 September 2000/Revised: 6 December 2000 相似文献
156.
157.
Electrophysiological investigations have been carried out to study the
uptake of nitrate and ammonium in the marine diatom Coscinodiscus
wailesii. Double-barrelled glass microelectrodes were used to
record the responses of voltage (V) and resistance
(R) between the inserted electrode tip and the outside
upon exposure to external nitrate or ammonium. Significant responses were
found to be restricted to certain periods following nitrogen-starvation,
but consistent with the requirements of nitrogen uptake under physiological
conditions. The response of V upon exposure to M concentrations of nitrate was a delayed and
reversible depolarization which decreased in amplitude upon repetitive
exposures in the time range of a minute. For the ionic conditions of
natural sea-water (about 500 mM Na+ and pH 8), these
effects are thermodynamically consistent with a sodium-nitrate symport
mechanism and inconsistent with the familiar proton-nitrate symport
mechanism in glycophytic cells of vascular plants and fresh water algae
(<1 mM Na+, pH <6). Replacement of
extracellular K+ in artificial sea-water (normal, 10
mM K+) by ammonium caused no significant
V response in non-starved cells; however,
depolarizations were observed in N-starved cells, indicating an increase of
the permeability ratio
Pammonium/Pk from about 1.0 to
about 1.2 under N-starvation. 相似文献
158.
159.
D M Gakamsky L F Boyd D H Margulies D M Davis J L Strominger I Pecht 《Biochemistry》1999,38(37):12165-12173
The mechanism of assembly/dissociation of a recombinant water-soluble class I major histocompatibility complex (MHC) H-2Kb molecule was studied by a real-time fluorescence resonance energy transfer method. Like the H-2Kd ternary complex [Gakamsky et al. (1996) Biochemistry 35, 14841-14848], the interactions among the heavy chain, beta2-microglobulin (beta2m), and antigenic peptides were found to be controlled by an allosteric mechanism. Association of the heavy chain with beta2m increased peptide binding rate constants by more than 2 orders of magnitude and enhanced affinity of the heavy-chain molecule for peptides. Interaction of peptides with the heavy-chain binding site, in turn, increased markedly the affinity of the heavy chain for beta2m. Binding of peptide variants of the ovalbumin sequence (257-264) to the heavy chain/beta2m heterodimer was found to be a biphasic reaction. The fast phase was a second-order process with nearly the same rate constants as those of binding of peptides derived from the influenza virus nucleoprotein 147-155 to the H-2Kd heavy chain/beta2m heterodimer [(3.0 +/- 1.0) x 10(-6) M-1 s-1 at 37 degrees C]. The slow phase was a result of both the ternary complex assembly from the "free" heavy chain, beta2m, and peptide as well as an intramolecular conformational transition within the heavy chain/beta2m heterodimer to a peptide binding conformation. Biexponential kinetics of peptide or beta2m dissociation from the ternary complex were observed. They suggest that it can exist in two conformations. The rate constants of beta2m dissociation from the H-2Kb ternary complex were, in the limits of experimental accuracy, independent of the structure of the bound peptide, though their affinities differed by an order of magnitude. Dissociation of peptides from the Kb heavy chain was always faster than from the ternary complexes, yet the heavy chain/peptide complexes were considerably more stable compared with their Kd/nucleoprotein peptide counterparts. 相似文献
160.