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81.
Photosynthetic O(2) production from water is catalyzed by a cluster of four manganese ions and a tyrosine residue that comprise the redox-active components of the water-oxidizing complex (WOC) of photosystem II (PSII) in all known oxygenic phototrophs. Knowledge of the oxidation states is indispensable for understanding the fundamental principles of catalysis by PSII and the catalytic mechanism of the WOC. Previous spectroscopic studies and redox titrations predicted the net oxidation state of the S(0) state to be (Mn(III))(3)Mn(IV). We have refined a previously developed photoassembly procedure that directly determines the number of oxidizing equivalents needed to assemble the Mn(4)Ca core of WOC during photoassembly, starting from free Mn(II) and the Mn-depleted apo-WOC complex. This experiment entails counting the number of light flashes required to produce the first O(2) molecules during photoassembly. Unlike spectroscopic methods, this process does not require reference to synthetic model complexes. We find the number of photoassembly intermediates required to reach the lowest oxidation state of the WOC, S(0), to be three, indicating a net oxidation state three equivalents above four Mn(II), formally (Mn(III))(3)Mn(II), whereas the O(2) releasing state, S(4), corresponds formally to (Mn(IV))(3)Mn(III). The results from this study have major implications for proposed mechanisms of photosynthetic water oxidation. 相似文献
82.
Débora Guerini Souza Bruna Bellaver Gisele Hansel Bernardo Assein Arús Gabriela Bellaver Aline Longoni Janaina Kolling Angela T. S. Wyse Diogo Onofre Souza André Quincozes-Santos 《Neurochemical research》2016,41(7):1578-1586
Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na+/K+-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate–glutamine cycle, as well as glutamate and d-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems. 相似文献
83.
Francieli M. Stefanello Cristiane Matté Carolina D. Pederzolli Janaína Kolling Caroline P. Mescka Marcelo L. Lamers Adriano M. de Assis Marcos L. Perry Marinilce F. dos Santos Carlos S. Dutra-Filho Angela T.S. Wyse 《Biochimie》2009
In the present study we evaluated the effect of chronic methionine administration on oxidative stress and biochemical parameters in liver and serum of rats, respectively. We also performed histological analysis in liver. Results showed that hypermethioninemia increased chemiluminescence, carbonyl content and glutathione peroxidase activity, decreased total antioxidant potential, as well as altered catalase activity. Hypermethioninemia increased synthesis and concentration of glycogen, besides histological studies showed morphological alterations and reduction in the glycogen/glycoprotein content in liver. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose were increased in hypermethioninemic rats. These findings suggest that oxidative damage and histological changes caused by methionine may be related to the hepatic injury observed in hypermethioninemia. 相似文献
84.
Niall G. Howlett Julie A. Harney Meghan A. Rego Frederick W. Kolling IV Thomas W. Glover 《The Journal of biological chemistry》2009,284(42):28935-28942
Fanconi Anemia (FA) is a rare recessive disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The FA proteins and the familial breast cancer susceptibility gene products, BRCA1 and FANCD1/BRCA2, function cooperatively in the FA-BRCA pathway to repair damaged DNA and to prevent cellular transformation. Activation of this pathway occurs via the mono-ubiquitination of the FANCD2 protein, targeting it to nuclear foci where it co-localizes with FANCD1/BRCA2, RAD51, and PCNA. The regulation of the mono-ubiquitination of FANCD2, as well as its function in DNA repair remain poorly understood. In this study, we have further characterized the interaction between the FANCD2 and PCNA proteins. We have identified a highly conserved, putative FANCD2 PCNA interaction motif (PIP-box), and demonstrate that mutation of this motif disrupts FANCD2-PCNA binding and precludes the mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant protein fails to correct the mitomycin C hypersensitivity of FA-D2 patient cells. Our results suggest that PCNA may function as a molecular platform to facilitate the mono-ubiquitination of FANCD2 and activation of the FA-BRCA pathway.Fanconi anemia (FA)2 is a rare recessive disorder characterized by developmental abnormalities, progressive bone marrow failure, and pronounced cancer susceptibility (1). FA patients are particularly susceptible to early-onset acute myelogenous leukemia and squamous cell carcinoma of the head, neck, and gynecologic regions (2). FA patient cells are hypersensitive to the clastogenic effects of DNA cross-linking agents, e.g. mitomycin C (MMC), and agents that inhibit DNA replication, e.g. aphidicolin (APH) (3, 4). There are currently thirteen genetically defined FA complementation groups (A, B, C, D1, D2, E, F, G, I, J, L, M, and N), and all thirteen genes have been identified (5).A central step in the activation of the FA-BRCA pathway is the mono-ubiquitination of the FANCD2 and FANCI proteins, catalyzed by the core FA E2/E3 holoenzyme complex (5, 6). The mono-ubiquitination of FANCD2 and FANCI signals their translocation to discrete nuclear foci, where they co-localize with the BRCA1 and RAD51 DNA repair proteins, as well as the major cellular DNA polymerase processivity factor PCNA (3, 4, 7–9). Several studies have suggested an important role for the FA-BRCA pathway in a DNA replication-associated DNA repair process, e.g. homologous recombination (HR), and/or translesion DNA synthesis (TLS) (3, 4, 10–12). Accordingly, additional proteins with established roles in the DNA replication stress response, including ATR, CHK1, HCLK2, and RPA, modulate DNA damage-inducible FANCD2 mono-ubiquitination (13–15). Our understanding of the regulation of this critical post-translational modification, however, is incomplete.We, and others (4, 7) have previously reported an association between FANCD2 and PCNA. FANCD2 and PCNA co-localize in nuclear foci following treatment with agents that inhibit DNA replication. Like FANCD2, PCNA is mono-ubiquitinated following exposure to DNA-damaging agents (16, 17). While FANCD2 and PCNA are mono-ubiquitinated by different E3 ubiquitin ligases, FANCL and RAD18 (16–19), respectively, both proteins are de-ubiquitinated by the USP1 enzyme (20, 21). The functional significance of the FANCD2-PCNA interaction, however, has not been determined.In addition to its role as a DNA polymerase processivity factor, PCNA interacts with many DNA repair proteins, e.g. MSH3, XPG, and p21Cip1/Waf1 (22). These interactions typically occur in a hydrophobic pocket of the PCNA homotrimer, termed the interdomain connecting loop (ICL). Proteins that interact with the PCNA ICL harbor a highly conserved PCNA-binding motif called the PIP-box, defined by the amino acid sequence QXXhXXaa, where h represents amino acids with moderately hydrophobic side chains, e.g. leucine, isoleucine, or methionine (L, I, M), a represents amino acids with highly hydrophobic, aromatic side chains, e.g. phenylalanine and tyrosine (F, Y), and X is any amino acid (23).Here, we describe an important functional interaction between FANCD2 and PCNA. We have identified a highly conserved putative PIP-box in FANCD2, and demonstrate that mutation of this motif disrupts the FANCD2-PCNA interaction, and precludes both the spontaneous and DNA damage-inducible mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant fails to correct the MMC hypersensitivity of FA-D2 patient-derived cells. However, the mutant protein retains the ability to localize to chromatin, interact with FANCE, and undergo DNA damage-inducible phosphorylation. Our results suggest that PCNA may act as a molecular platform for the mono-ubiquitination of FANCD2 and for the activation of the FA-BRCA pathway. 相似文献
85.
Deise Juliana Kolling Jean Borges Bertoldo Fábio Cristiano Angonesi Brod Javier Vernal Hernán Terenzi Ana Carolina Maisonnave Arisi 《Molecular biotechnology》2010,46(2):168-175
Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific
activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis.
Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity
was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp
presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature
(pH 9.0 and 42°C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80°C all
enzymes retained their initial activities. 相似文献
86.
87.
A model of the carbohydrate recognition domain CRD, residues 111-245, of
hamster galectin-3 has been made using homology modeling and dynamics
minimization methods. The model is based on the known x-ray structures of
bovine galectin-1 and human galectin-2. The oligosaccharides
NeuNAc-alpha2,3-Gal-beta1,4-Glc and GalNAc-alpha1, 3-
[Fuc-alpha1,2]-Gal-beta1,4-Glc, known to be specific high-affinity ligands
for galectin-3, as well as lactose recognized by all galectins were docked
in the galectin-3 CRD model structure and a minimized binding conformation
found in each case. These studies indicate a putative extended
carbohydrate-binding subsite in the hamster galectin- 3 involving Arg139,
Glu230, and Ser232 for NeuNAc-alpha2,3-; Arg139 and Glu160 for
fucose-alpha1,2-; and Arg139 and Ile141 for GalNAc-alpha1,3- substituents
on the primary galactose. Each of these positions is variable within the
whole galectin family. Two of these residues, Arg139 and Ser232, were
selected for mutagenesis to probe their importance in this newly identified
putative subsite. Residue 139 adopts main-chain dihedral angles
characteristic of an isolated bridge structural feature, while residue 232
is the C-terminal residue of beta- strand-11, and is followed immediately
by an inverse gamma-turn. A systematic series of mutant proteins have been
prepared to represent the residue variation present in the aligned
sequences of galectins-1, - 2, and -3. Minimized docked models were
generated for each mutant in complex with NeuNAc-alpha2,3-Gal-beta1,4-Glc,
GalNAc-alpha1, 3-[Fuc- alpha1,2]-Gal-beta1,4- Glc, and Gal-beta1,4-Glc.
Correlation of the computed protein-carbohydrate interaction energies for
each lectin- oligosaccharide pair with the experimentally determined
binding affinities for fetuin and asialofetuin or the relative potencies of
lactose and sialyllactose in inhibiting binding to asiolofetuin is
consistent with the postulated key importance of Arg139 in recognition of
the extended sialylated ligand.
相似文献
88.
Nucleotide sequence analysis of the human salivary protein genes HIS1 and HIS2, and evolution of the STATH/HIS gene family 总被引:1,自引:0,他引:1
Human histatins are a family of low-M(r), neutral to very basic,
histidine-rich salivary polypeptides. They probably function as part of the
nonimmune host defense system in the oral cavity. A 39-kb region of DNA
containing the HIS1 and HIS2 genes was isolated from two human genomic
phage libraries as a series of overlapping clones. The nucleotide sequences
of the HIS1 gene and part of the HIS2(1) gene were determined. The
transcribed region of HIS1 spans 8.5 kb and contains six exons and five
introns. The HIS1 and HIS2(1) genes exhibit 89% overall sequence identity,
with exon sequences exhibiting 95% identity. The two loci probably arose by
a gene duplication event approximately 15-30 Mya. The HIS1 sequence data
were also compared with that of STATH. Human statherin is a low-M(r) acidic
phosphoprotein that acts as an inhibitor of precipitation of calcium
phosphate salts in the oral cavity. The HIS1 and STATH genes show nearly
identical overall gene structures. The HIS1 and STATH loci exhibit 77%-81%
sequence identity in intron DNA and 80%-88% sequence identity in noncoding
exons but only 38%-43% sequence identity in the protein-coding regions of
exons 4 and 5. These unusual data suggest that HIS1, HIS2, and STATH belong
to a single gene family exhibiting accelerated evolution between the HIS
and STATH coding sequences.
相似文献
89.
Evolution of the Adh locus in the Drosophila willistoni group: the loss of an intron, and shift in codon usage 总被引:1,自引:0,他引:1
We report here the DNA sequence of the alcohol dehydrogenase gene (Adh)
cloned from Drosophila willistoni. The three major findings are as follows:
(1) Relative to all other Adh genes known from Drosophila, D. willistoni
Adh has the last intron precisely deleted; PCR directly from total genomic
DNA indicates that the deletion exists in all members of the willistoni
group but not in any other group, including the closely related saltans
group. Otherwise the structure and predicted protein are very similar to
those of other species. (2) There is a significant shift in codon usage,
especially compared with that in D. melanogaster Adh. The most striking
shift is from C to U in the wobble position (both third and first
position). Unlike the codon-usage-bias pattern typical of highly biased
genes in D. melanogaster, including Adh, D. willistoni has nearly 50% G + C
in the third position. (3) The phylogenetic information provided by this
new sequence is in agreement with almost all other molecular and
morphological data, in placing the obscura group closer to the melanogaster
group, with the willistoni group farther distant but still clearly within
the subgenus Sophophora.
相似文献
90.
An exception to the generally conservative nature of plastid gene evolution
is the gene coding for the beta" subunit of RNA polymerase, rpoC2. Previous
work by others has shown that maize and rice have an insertion in the
coding region of rpoC2, relative to spinach and tobacco. To assess the
distribution of this extra coding sequence, we surveyed a broad
phylogenetic sample comprising 55 species from 17 angiosperm families by
using Southern hybridization. The extra coding sequence is restricted to
the grasses (Poaceae). DNA sequence analysis of 11 species from all five
subfamilies within the grass family demonstrates that the extra sequence in
the coding region of rpoC2 is a repetitive array that exhibits more than a
twofold increase in nucleotide substitution, as well as a large number of
insertion/deletion events, relative to the adjacent flanking sequences. The
structure of the array suggests that slipped-strand mispairing causes the
repeated motifs and adds to the mechanisms through which the coding
sequence of plastid genes are known to evolve. Phylogenetic analyses based
on the sequence data from grass species support several relationships
previously suggested by morphological work, but they are ambiguous about
broad relationships within the family.
相似文献