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71.
The nematode parasites Wuchereria bancrofti, Brugia malayi, and B. timori cause a disease in humans known as lymphatic filariasis, which afflicts approximately 120 million people worldwide. The parasites enter the human host from the mosquito either as L3 or as infective larvae and subsequently differentiate through 2 molts. In this article, we show that B. malayi depends on an exogenous source of vitamin C to complete the L3 to L4 molt, a critical morphogenic step in its life cycle. Brugia malayi apparently belongs to a small group of living organisms that depend on an exogenous source of vitamin C. This group includes only primates (including man) and guinea pigs among mammals. 相似文献
72.
Three-dimensional localization of pORF65 in Kaposi's sarcoma-associated herpesvirus capsid 下载免费PDF全文
Of the six herpesvirus capsid proteins, the smallest capsid proteins (SCPs) share the least sequence homology among herpesvirus family members and have been implicated in virus specificity during infection. The herpes simplex virus-1 (HSV-1) SCP was shown to be horn shaped and to specifically bind the upper domain of each major capsid protein in hexons but not in pentons. In Kaposi's sarcoma-associated herpesvirus (KSHV), the protein encoded by the ORF65 gene (pORF65) is the putative SCP but its location remains controversial due to the absence of such horn-shaped densities from both the pentons and hexons of the KSHV capsid reconstructions. To directly locate the KSHV SCP, we have used electron cryomicroscopy and three-dimensional reconstruction techniques to compare the three-dimensional structure of KSHV capsids to that of anti-pORF65 antibody-labeled capsids. Our difference map shows prominent antibody densities bound to the tips of the hexons but not to pentons, indicating that KSHV SCP is attached to the upper domain of the major capsid protein in hexons but not to that in pentons, similar to HSV-1 SCP. The lack of horn-shaped densities on the hexons indicates that KSHV SCP exhibits structural features that are substantially different from those of HSV-1 SCP. The location of SCP at the outermost regions of the capsid suggests a possible role in mediating capsid interactions with the tegument and cytoskeletal proteins during infection. 相似文献
73.
Three-dimensional structures of the A, B, and C capsids of rhesus monkey rhadinovirus: insights into gammaherpesvirus capsid assembly, maturation, and DNA packaging 下载免费PDF全文
Yu XK O'Connor CM Atanasov I Damania B Kedes DH Zhou ZH 《Journal of virology》2003,77(24):13182-13193
Rhesus monkey rhadinovirus (RRV) exhibits high levels of sequence homology to human gammaherpesviruses, such as Kaposi's sarcoma-associated herpesvirus, and grows to high titers in cell cultures, making it a good model system for studying gammaherpesvirus capsid structure and assembly. We have purified RRV A, B, and C capsids, thus for the first time allowing direct structure comparisons by electron cryomicroscopy and three-dimensional reconstruction. The results show that the shells of these capsids are identical and are each composed of 12 pentons, 150 hexons, and 320 triplexes. Structural differences were apparent inside the shells and through the penton channels. The A capsid is empty, and its penton channels are open. The B capsid contains a scaffolding core, and its penton channels are closed. The C capsid contains a DNA genome, which is closely packaged into regularly spaced density shells (25 A apart), and its penton channels are open. The different statuses of the penton channels suggest a functional role of the channels during capsid maturation, and the overall structural similarities of RRV capsids to alphaherpesvirus capsids suggest a common assembly and maturation pathway. The RRV A capsid reconstruction at a 15-A resolution, the best achieved for gammaherpesvirus particles, reveals overall structural similarities to alpha- and betaherpesvirus capsids. However, the outer regions of the capsid, including densities attributed to the Ta triplex and the small capsomer-interacting protein (SCIP or ORF65), exhibit prominent differences from their structural counterparts in alphaherpesviruses. This structural disparity suggests that SCIP and the triplex, together with tegument and envelope proteins, confer structural and potentially functional specificities to alpha-, beta-, and gammaherpesviruses. 相似文献
74.
Primary reactions of the LOV2 domain of phototropin,a plant blue-light photoreceptor 总被引:4,自引:0,他引:4
Kennis JT Crosson S Gauden M van Stokkum IH Moffat K van Grondelle R 《Biochemistry》2003,42(12):3385-3392
The phototropins constitute an important class of plant photoreceptor kinases that control a range of physiological responses, including phototropism, light-directed chloroplast movement, and light-induced stomatal opening. The LOV2 domain of phototropin binds a molecule of flavin mononucleotide (FMN) and undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine residue and the C(4a) atom of FMN. This product state promotes C-terminal kinase activation and downstream signal transduction. Here, we report the primary photophysics and photochemistry of LOV2 domains of phototropin 1 of Avena sativa (oat) and of the phy3 photoreceptor of Adiantum capillus-veneris (maidenhair fern). In agreement with earlier reports [Swartz, T. E., et al. (2001) J. Biol. Chem. 276, 36493-36500], we find that the FMN triplet state is the reactive species from which the photoreaction occurs. We demonstrate that the triplet state is the primary photoproduct in the LOV2 photocycle, generated at 60% efficiency. No spectroscopically distinguishable intermediates precede the FMN triplet on the femtosecond to nanosecond time scale, indicating that it is formed directly via intersystem crossing (ISC) from the singlet state. Our results indicate that the majority of the FMN triplets in the LOV2 domain exist in the protonated form. We propose a reaction mechanism that involves excited-state proton transfer, on the nanosecond time scale or faster, from the sulfhydryl group of the conserved cysteine to the N5 atom of FMN. This event promotes adduct formation by increasing the electrophilicity of C(4a) and subsequent nucleophilic attack by the cysteine's thiolate anion. Comparison to free FMN in solution shows that the protein environment of LOV2 increases the ISC rate of FMN by a factor of 2.4, thus improving the yield of the cysteinyl-flavin adduct and the efficiency of phototropin-mediated signaling processes. 相似文献
75.
Haker A Hendriks J van Stokkum IH Heberle J Hellingwerf KJ Crielaard W Gensch T 《The Journal of biological chemistry》2003,278(10):8442-8451
The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP(360)) and 446 nm (R-PYP(446)), respectively. Both forms are photoactive and part of a temperature- and pH-dependent equilibrium (Haker, A., Hendriks, J., Gensch, T., Hellingwerf, K. J., and Crielaard, W. (2000) FEBS Lett. 486, 52-56). At 20 degrees C, for PYP characteristic, the 446-nm absorbance band displays a photocycle, in which the depletion of the 446-nm ground state absorption occurs in at least three phases, with time constants of <30 ns, 0.5 micros, and 17 micros. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB(360), lambda(max) = 360 nm) is established. The photocycle is completed with a monophasic recovery of the ground state with a time constant of 2.5 ms. At 7 degrees C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP(360) with a UV-flash (330 +/- 50 nm) a species with a difference absorption maximum at approximately 435 nm is observed that returns to R-PYP(360) on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP(360) and R-PYP(446) differ in their overall protein conformation, as well as in the isomerization and protonation state of the chromophore, as determined with the fluorescent polarity probe Nile Red and Fourier Transform Infrared spectroscopy, respectively. 相似文献
76.
Ivo Šafařík Pavel Mucha Jiří Pechoč Jaroslav Stoklasa Mirka Šafaříková 《Biotechnology letters》2001,23(11):851-855
A Davis tube (a matrix-free, flow-through magnetic separator used mainly in mineral processing) has been tested for separation of magnetic affinity biopolymer adsorbents from larger volumes of suspensions. Both magnetic chitosan and magnetic cross-linked erythrocytes could be efficiently separated from litre volumes of suspensions. Up to 90% adsorbent recovery was achieved under optimised separation conditions. 相似文献
77.
Large-scale separation of magnetic bioaffinity adsorbents 总被引:1,自引:0,他引:1
Flat magnetic separator was used to separate magnetic bioaffinity adsorbents from litre volumes of suspensions. Both magnetic cross-linked erythrocytes and magnetic chitosan were efficiently separated; at least 95% adsorbent recovery was achieved at maximum flow rate (1680 ml min–1). Using this system low amounts of trypsin were concentrated from large sample volumes using magnetic erythrocytes as affinity adsorbent. 相似文献
78.
79.
Cytokinin oxidase/cytokinin dehydrogenase assay: optimized procedures and applications 总被引:1,自引:0,他引:1
1H NMR spectroscopy has been used to assess long-term toxicological effects of a rare earth. Male Wistar rats were administrated orally with La(NO3)3 at doses of 0.1, 0.2, 2.0, 10, and 20 mg/kg body wt, resp., for 3-6 months. Urine was collected at 1, 2, and 3 months and serum samples were taken after 6 months. Numerous low-M(r) metabolites in rats serum and rats urine, including creatinine, citrate, glucose, ketone bodies, trimethylamine N-oxide (TMAO), and various amino acids, were identified on 400- and 500-MHz 1H NMR spectra. La3+-induced renal and liver damage is characterized by an increase in the amounts of the excreted ketone bodies, amino acids, lactate, ethanol, succinate, TMAO, dimethylamine, and taurine and a decrease in citrate, glucose, urea, and allantoin. Information on the molecular basis of the long-term toxicity of La(NO>3)3 was derived from the abnormal patterns of metabolite excretions. An assay of some biochemical indexes and analysis of some enzymes in plasma supported NMR results. 相似文献
80.
Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum 总被引:4,自引:0,他引:4 下载免费PDF全文
Flavivirus envelope proteins are synthesized as part of large polyproteins that are co- and posttranslationally cleaved into their individual chains. To investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (TBE) virus envelope glycoproteins prM and E by using a recombinant plasmid expression system and virus-infected cells. When expressed in their polyprotein context, prM and E achieved their native folded structures with half-times of approximately 4 min for prM and about 15 min for E. They formed heterodimeric complexes within a few minutes after synthesis that were required for the final folding of E but not for that of prM. Heterodimers could also be formed in trans when these proteins were coexpressed from separate constructs. When expressed without prM, E could form disulfide bonds but did not express a specific conformational epitope and remained sensitive to reduction by dithiothreitol. This is consistent with a chaperone-like role for prM in the folding of E. PrM was able to achieve its native folded structure without coexpression of E, but signal sequence cleavage at the N terminus was delayed. Our results show that prM is an especially rapidly folding viral glycoprotein, that polyprotein cleavage and folding of the TBE virus envelope proteins occurs in a coordinated sequence of processing steps, and that proper and efficient maturation of prM and E can only be achieved by cosynthesis of these two proteins. 相似文献