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211.
Cholesterol is known to play an important role in stabilizing particular cellular membrane structures, so-called lipid or membrane rafts. For several viruses, a dependence on cholesterol for virus entry and/or morphogenesis has been shown. Using flow cytometry and fluorescence microscopy, we demonstrate that infection of cells by canine distemper virus (CDV) was not impaired after cellular cholesterol had been depleted by the drug methyl-beta-cyclodextrin. This effect was independent of the multiplicity of infection and the cellular receptor used for infection. However, cholesterol depletion of the viral envelope significantly reduced CDV infectivity. Replenishment by addition of exogenous cholesterol restored infectivity up to 80%. Thus, we conclude that CDV entry is dependent on cholesterol in the viral envelope. Furthermore, reduced syncytium formation was observed when the cells were cholesterol depleted during the course of the infection. This may be related to the observation that CDV envelope proteins H and F partitioned into cellular detergent-resistant membranes. Therefore, a role for lipid rafts during virus assembly and release as well is suggested. 相似文献
212.
Expression and characterization of a minimal hepatitis C virus glycoprotein E2 core domain that retains CD81 binding
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The hepatitis C virus glycoprotein E2 receptor-binding domain is encompassed by amino acids 384 to 661 (E2(661)) and contains two hypervariable sequences, HVR1 and HVR2. E2 sequence comparisons revealed a third variable region, located between residues 570 and 580, that varies widely between genotypes, designated here as igVR, the intergenotypic variable region. A secreted E2(661) glycoprotein with simultaneous deletions of the three variable sequences retained its ability to bind CD81 and conformation-dependent monoclonal antibodies (MAbs) and displayed enhanced binding to a neutralizing MAb directed to E2 immunogenic domain B. Our data provide insights into the E2 structure by suggesting that the three variable regions reside outside a conserved E2 core. 相似文献
213.
A capillary electrophoresis method to assay catalytic activity of botulinum neurotoxin serotypes: implications for substrate specificity 总被引:1,自引:0,他引:1
The potent botulinum neurotoxin inhibits neurotransmitter release at cholinergic nerve terminals, causing a descending flaccid paralysis characteristic of the disease botulism. The currently expanding medical use of the neurotoxin to treat several disorders, as well as the potential misuse of the neurotoxin as an agent in biowarfare, has made understanding of the nature of the toxin's catalytic activity and development of inhibitors critical. To study the catalytic activity of botulinum neurotoxin more thoroughly and characterize potential inhibitors, we have developed a capillary electrophoresis method to measure catalytic activity of different serotypes of botulinum neurotoxin using peptides derived from the native substrates. This assay requires only a minute amount of sample (25 nl), is relatively rapid (15 min/sample), and allows the determination of enzyme kinetic constants for a more sophisticated characterization of inhibitors and neurotoxin catalytic activity. Using this method, we can measure activity of five of the seven serotypes of botulinum neurotoxin (A, B, E, F, and G) with two peptide substrates. Botulinum neurotoxin serotypes C and D did not cleave our peptides, lending insight into potential substrate requirements among the serotypes. 相似文献
214.
Jämsen J Tuominen H Salminen A Belogurov GA Magretova NN Baykov AA Lahti R 《The Biochemical journal》2007,408(3):327-333
CBS (cystathionine beta-synthase) domains are found in proteins from all kingdoms of life, and point mutations in these domains are responsible for a variety of hereditary diseases in humans; however, the functions of CBS domains are not well understood. In the present study, we cloned, expressed in Escherichia coli, and characterized a family II PPase (inorganic pyrophosphatase) from Moorella thermoacetica (mtCBS-PPase) that has a pair of tandem 60-amino-acid CBS domains within its N-terminal domain. Because mtCBS-PPase is a dimer and requires transition metal ions (Co2+ or Mn2+) for activity, it resembles common family II PPases, which lack CBS domains. The mtCBS-PPase, however, has lower activity than common family II PPases, is potently inhibited by ADP and AMP, and is activated up to 1.6-fold by ATP. Inhibition by AMP is competitive, whereas inhibition by ADP and activation by ATP are both of mixed types. The nucleotides are effective at nanomolar (ADP) or micromolar concentrations (AMP and ATP) and appear to compete for the same site on the enzyme. The nucleotide-binding affinities are thus 100-10000-fold higher than for other CBS-domain-containing proteins. Interestingly, genes encoding CBS-PPase occur most frequently in bacteria that have a membrane-bound H+-translocating PPase with a comparable PP(i)-hydrolysing activity. Our results suggest that soluble nucleotide-regulated PPases act as amplifiers of metabolism in bacteria by enhancing or suppressing ATP production and biosynthetic reactions at high and low [ATP]/([AMP]+[ADP]) ratios respectively. 相似文献
215.
LRIG1 is a novel negative regulator of the Met receptor and opposes Met and Her2 synergy 总被引:5,自引:0,他引:5
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Shattuck DL Miller JK Laederich M Funes M Petersen H Carraway KL Sweeney C 《Molecular and cellular biology》2007,27(5):1934-1946
The Met receptor tyrosine kinase regulates a complex array of cellular behaviors collectively known as "invasive growth." While essential for normal development and wound repair, this program is frequently co-opted by tumors to promote their own growth, motility, and invasion. Met is overexpressed in a variety of human tumors, and this aberrant expression correlates with poor patient prognosis. Previous studies indicate that Met receptor levels are governed in part by cbl-mediated ubiquitination and degradation, and uncoupling of Met from cbl-mediated ubiquitination promotes its transforming activity. Here we describe a novel mechanism for Met degradation. We find that the Met receptor interacts with the transmembrane protein LRIG1 independent of hepatocyte growth factor (HGF) stimulation and that LRIG1 destabilizes the Met receptor in a cbl-independent manner. Overexpression of LRIG1 destabilizes endogenous Met receptor in breast cancer cells and impairs their ability to respond to HGF. LRIG1 knockdown increases Met receptor half-life, indicating that it plays an essential role in Met degradation. Finally, LRIG1 opposes Met synergy with the ErbB2/Her2 receptor tyrosine kinase in driving cellular invasion. We conclude that LRIG1 is a novel suppressor of Met function, serving to regulate cellular receptor levels by promoting Met degradation in a ligand- and cbl-independent manner. 相似文献
216.
Groome JR Alexander HM Fujimoto E Sherry M Petty D 《Cellular and molecular neurobiology》2007,27(1):87-106
Summary 1. Mutations in the S4 segment of domain III in the voltage gated skeletal muscle sodium channel hNaV1.4 were constructed to test the roles of each charged residue in deactivation gating. Mutations comprised charge reversals
at K1-R6, charge neutralization, and substitution at R4 and R5.
2. Charge-reversing mutations at R4 and R5 produced the greatest alteration of activation parameters compared to hNaV1.4. Effects included depolarization of the conductance/voltage (g/V) curve, decreased valence and slowing of kinetics.
3. Reversal of charge at R2 to R4 hyperpolarized, and reversal at R5 or R6 depolarized the h
∞ curve. Most DIIIS4 mutations slowed inactivation from the open state. R4E slowed closed state fast inactivation and R5E inhibited
its completion.
4. Deactivation from the open and/or inactivated state was prolonged in mutations reversing charge at R2 to R4 but accelerated
by reversal of charge at R5 or R6. Effects were most pronounced at central charges R4 and R5.
5. Charge and structure each contribute to effects of mutations at R4 and R5 on channel gating. Effects of mutations on activation
and deactivation at R4 and, to a lesser extent R5, were primarily owing to charge alteration, whereas effects on fast inactivation
were charge independent. 相似文献
217.
218.
A conventional extraction technique of sonication has been compared, in terms of extraction efficiency, extraction time and amount of solvent, with the more novel technique of accelerated solvent extraction for the extraction of kavain from the powdered roots of Piper methysticum (Kava) with acetone. The extracts were analysed using high-performance liquid chromatography with ultra violet detection. The effects of varying solvent volume and extraction time upon the quantity of kavain extracted with sonication, and the effects of varying temperature upon the kavain extraction efficiency by ASE, were investigated. ASE was found to be more efficient with respect to time and solvent volume required; however, a good agreement was found between the kavain concentration obtained using both extraction techniques. 相似文献
219.
Roggen H Kehler J Stensbøl TB Hansen T 《Bioorganic & medicinal chemistry letters》2007,17(10):2834-2837
A series of Milnacipran analogs with variation in the aromatic moiety were prepared in high enantiomeric excess. Structure-activity relationships for two parallel enantiomeric series are described. The (-)-(1R,2S)-naphthyl analog (8h) showed the highest potency in the two series and is a triple reuptake inhibitor of the SERT, NET, and DAT. 相似文献