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1.
《The Journal of biological chemistry》2014,289(26):17970
2.
Xing Chen Feng Gao Wei‐Yan Yang Zhu‐Xin Zhou Jin‐Qiang Lin Liang‐Nian Ji 《化学与生物多样性》2013,10(3):367-384
To investigate the relationship between the molecular structure and biological activity of polypyridyl RuII complexes, such as DNA binding, photocleavage ability, and DNA topoisomerase and RNA polymerase inhibition, six new [Ru(bpy)2(dppz)]2+ (bpy=2,2′‐bipyridine; dppz=dipyrido[3,2‐a:2,′,3′‐c]phenazine) analogs have been synthesized and characterized by means of 1H‐NMR spectroscopy, mass spectrometry, and elemental analysis. Interestingly, the biological properties of these complexes have been identified to be quite different via a series of experimental methods, such as spectral titration, DNA thermal denaturation, viscosity, and gel electrophoresis. To explain the experimental regularity and reveal the underlying mechanism of biological activity, the properties of energy levels and population of frontier molecular orbitals and excited‐state transitions of these complexes have been studied by density‐functional theory (DFT) and time‐depended DFT (TDDFT) calculations. The results suggest that DNA intercalative ligands with better planarity, greater hydrophobicity, and less steric hindrance are beneficial to the DNA intercalation and enzymatic inhibition of their complexes. 相似文献
3.
The action of the environmental toxic Pb2+ on photosynthetic electron transport was studied in thylakoid membranes isolated from spinach leaves. Fluorescence and thermoluminescence techniques were performed in order to determine the mode of Pb2+ action in photosystem II (PSII). The invariance of fluorescence characteristics of chlorophyll a (Chl a) and magnesium tetraphenylporphyrin (MgTPP), a molecule structurally analogous to Chl a, in the presence of Pb2+ confirms that Pb cation does not interact directly with chlorophyll molecules in PSII. The results show that Pb interacts with the water oxidation complex thus perturbing charge recombination between the quinone acceptors of PSII and the S2 state of the Mn4Ca cluster. Electron transfer between the quinone acceptors QA and QB is also greatly retarded in the presence of Pb2+. This is proposed to be owing to a transmembrane modification of the acceptor side of the photosystem. 相似文献
4.
Lisa Placanica Leonid Tarassishin Guangli Yang Erica Peethumnongsin Seong-Hun Kim Hui Zheng Sangram S. Sisodia Yue-Ming Li 《The Journal of biological chemistry》2009,284(5):2967-2977
γ-Secretase is known to play a pivotal role in the pathogenesis of
Alzheimer disease through production of amyloidogenic Aβ42 peptides.
Early onset familial Alzheimer disease mutations in presenilin (PS), the
catalytic core of γ-secretase, invariably increase the
Aβ42:Aβ40 ratio. However, the mechanism by which these mutations
affect γ-secretase complex formation and cleavage specificity is poorly
understood. We show that our in vitro assay system recapitulates the
effect of PS1 mutations on the Aβ42:Aβ40 ratio observed in cell and
animal models. We have developed a series of small molecule affinity probes
that allow us to characterize active γ-secretase complexes. Furthermore
we reveal that the equilibrium of PS1- and PS2-containing active complexes is
dynamic and altered by overexpression of Pen2 or PS1 mutants and that
formation of PS2 complexes is positively correlated with increased
Aβ42:Aβ40 ratios. These data suggest that perturbations to
γ-secretase complex equilibrium can have a profound effect on enzyme
activity and that increased PS2 complexes along with mutated PS1 complexes
contribute to an increased Aβ42:Aβ40 ratio.β-Amyloid
(Aβ)5 peptides
are believed to play a causative role in Alzheimer disease (AD). Aβ
peptides are generated from the processing of the amyloid precursor protein
(APP) by two proteases, β-secretase and γ-secretase. Although
γ-secretase generates heterogenous Aβ peptides ranging from 37 to
46 amino acids in length, significant work has focused mainly on the Aβ40
and Aβ42 peptides that are the major constituents of amyloid plaques.
γ-Secretase is a multisubunit membrane aspartyl protease comprised of at
least four known subunits: presenilin (PS), nicastrin (Nct), anterior
pharynx-defective (Aph), and presenilin enhancer 2 (Pen2). Presenilin is
thought to contain the catalytic core of the complex
(1–4),
whereas Aph and Nct play critical roles in the assembly, trafficking, and
stability of γ-secretase as well as substrate recognition
(5,
6). Lastly Pen2 facilitates the
endoproteolysis of PS into its N-terminal (NTF) and C-terminal (CTF) fragments
thereby yielding a catalytically competent enzyme
(5,
7–10).
All four proteins (PS, Nct, Aph1, and Pen2) are obligatory for
γ-secretase activity in cell and animal models
(11,
12). There are two homologs of
PS, PS1 and PS2, and three isoforms of Aph1, Aph1aS, Aph1aL, and Aph1b. At
least six active γ-secretase complexes have been reported (two
presenilins × three Aph1s)
(13,
14). The sum of apparent
molecular masses of the four proteins (PS1-NTF/CTF ≈ 53 kDa, Nct ≈ 120
kDa, Aph1 ≈ 30 kDa, and Pen2 ≈ 10kDa) is ∼200 kDa. However, active
γ-secretase complexes of varying sizes, ranging from 250 to 2000 kDa,
have been reported
(15–19).
Recently a study suggested that the γ-secretase complex contains only
one of each subunit (20).
Collectively these studies suggest that a four-protein complex around
200–250 kDa may be the minimal functional γ-secretase unit with
additional cofactors and/or varying stoichiometry of subunits existing in the
high molecular weight γ-secretase complexes. CD147 and TMP21 have been
found to be associated with the γ-secretase complex
(21,
22); however, their role in
the regulation of γ-secretase has been controversial
(23,
24).Mutations of PS1 or PS2 are associated with familial early onset AD (FAD),
although it is debatable whether these familial PS mutations act as
“gain or loss of function” alterations in regard to
γ-secretase activity
(25–27).
Regardless the overall outcome of these mutations is an increased ratio of
Aβ42:Aβ40. Clearly these mutations differentially affect
γ-secretase activity for the production of Aβ40 and Aβ42.
Despite intensive studies of Aβ peptides and γ-secretase, the
molecular mechanism controlling the specificity of γ-secretase activity
for Aβ40 and Aβ42 production has not been resolved. It has been
found that PS1 mutations affect the formation of γ-secretase complexes
(28). However, the precise
mechanism by which individual subunits alter the dynamics of γ-secretase
complex formation and activity is largely unresolved. A better mechanistic
understanding of γ-secretase activity associated with FAD mutations has
been hindered by the lack of suitable assays and probes that are necessary to
recapitulate the effect of these mutations seen in cell models and to
characterize the active γ-secretase complex.In our present studies, we have determined the overall effect of Pen2 and
PS1 expression on the dynamics of PS1- and PS2-containing complexes and their
association with γ-secretase activity. Using newly developed
biotinylated small molecular probes and activity assays, we revealed that
expression of Pen2 or PS1 FAD mutants markedly shifts the equilibrium of
PS1-containing active complexes to that of PS2-containing complexes and
results in an overall increase in the Aβ42:Aβ40 ratio in both stable
cell lines and animal models. Our studies indicate that perturbations to the
equilibrium of active γ-secretase complexes by an individual subunit can
greatly affect the activity of the enzyme. Moreover they serve as further
evidence that there are multiple and distinct γ-secretase complexes that
can exist within the same cells and that their equilibrium is dynamic.
Additionally the affinity probes developed here will facilitate further study
of the expression and composition of endogenous active γ-secretase from
a variety of model systems. 相似文献
5.
Fernando Gómez-Herreros Rocío Romero-Granados Zhihong Zeng Alejandro álvarez-Quilón Cristina Quintero Limei Ju Lieve Umans Liesbeth Vermeire Danny Huylebroeck Keith W. Caldecott Felipe Cortés-Ledesma 《PLoS genetics》2013,9(3)
Anticancer topoisomerase “poisons” exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5′-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy. 相似文献
6.
Cell death can be divided into the anti-inflammatory process of apoptosis and the
pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell
death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP)
1/3 are major mediators. We previously showed that absence or inhibition of PARP-1
protects mice from nephritis, however only the male mice. We therefore hypothesized that
there is an inherent difference in the cell death program between the sexes. We show here
that in an immune-mediated nephritis model, female mice show increased apoptosis compared
to male mice. Treatment of the male mice with estrogens induced apoptosis to levels
similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in
both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We
also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male
and female mice are prone to different types of cell death. Our data also suggest that
estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore
propose that targeting cell death based on sex will lead to tailored and better treatments
for each gender. 相似文献
7.
8.
Z. A. Sergeeva A. G. Venyaminova V. F. Zarytova 《Nucleosides, nucleotides & nucleic acids》2013,32(9-11):2153-2156
Abstract The results of modification of the model DNA and RNA targets by the alkylating derivatives of 2′-O-methylribo-, ribo-, and deoxyhexanucleotides in the presence and absence of effectors (N-(2-hydroxyethyl)phenazinium derivatives of the same type of octanucleotides) are presented. It has been shown that the alkylating 4(N-methyl-N-2-chloroethyl-)benzylmethylamidophosphate derivatives of oligo(2′-O-methylribonucleotides) are the high effective reagents for the site specific modification of nucleic acids especially RNA. 相似文献
9.
Giovanni Maga Barbara van Loon Emmanuele Crespan Giuseppe Villani Ulrich H��bscher 《The Journal of biological chemistry》2009,284(21):14267-14275
Abasic (AP) sites are very frequent and dangerous DNA lesions. Their
ability to block the advancement of a replication fork has been always viewed
as a consequence of their inhibitory effect on the DNA synthetic activity of
replicative DNA polymerases (DNA pols). Here we show that AP sites can also
affect the strand displacement activity of the lagging strand DNA pol δ,
thus preventing proper Okazaki fragment maturation. This block can be overcome
through a polymerase switch, involving the combined physical and functional
interaction of DNA pol β and Flap endonuclease 1. Our data identify a
previously unnoticed deleterious effect of the AP site lesion on normal cell
metabolism and suggest the existence of a novel repair pathway that might be
important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage
in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic)
lesions occur spontaneously in DNA; in eukaryotes it has been estimated that
about 104 depurination and 102 depyrimidation events
occur per genome per day. An equally important source of abasic DNA lesions
results from the action of DNA glycosylases, such as uracil glycosylase, which
excises uracil arising primarily from spontaneous deamination of cytosines
(1). Although most AP sites are
removed by the base excision repair
(BER)5 pathway, a
small fraction of lesions persists, and DNA with AP lesions presents a strong
block to DNA synthesis by replicative DNA polymerases (DNA pols)
(2,
3). Several studies have been
performed to address the effects of AP sites on the template DNA strand on the
synthetic activity of a variety of DNA pols. The major replicative enzyme of
eukaryotic cells, DNA pol δ, was shown to be able to bypass an AP
lesion, but only in the presence of the auxiliary factor proliferating cell
nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared
with an undamaged DNA template
(4). On the other hand, the
family X DNA pols β and λ were shown to bypass an AP site but in a
very mutagenic way (5). Recent
genetic evidence in Saccharomyces cerevisiae cells showed that DNA
pol δ is the enzyme replicating the lagging strand
(6). According to the current
model for Okazaki fragment synthesis
(7–9),
the action of DNA pol δ is not only critical for the extension of the
newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA
segment of about 30 nucleotides on the pre-existing downstream Okazaki
fragment to create an intermediate Flap structure that is the target for the
subsequent action of the Dna2 endonuclease and the Flap endonuclease 1
(Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid
fragment synthesized by the DNA pol α/primase, potentially containing
nucleotide misincorporations caused by the lack of a proofreading exonuclease
activity of DNA pol α/primase. This results in a more accurate copy
synthesized by DNA pol δ. The intrinsic strand displacement activity of
DNA pol δ, in conjunction with Fen-1, PCNA, and replication protein A
(RP-A), has been also proposed to be essential for the S phase-specific long
patch BER pathway (10,
11). Although it is clear that
an AP site on the template strand is a strong block for DNA pol
δ-dependent synthesis on single-stranded DNA, the functional
consequences of such a lesion on the ability of DNA pol δ to carry on
strand displacement synthesis have never been investigated so far. Given the
high frequency of spontaneous hydrolysis and/or cytidine deamination events,
any detrimental effect of an AP site on the strand displacement activity of
DNA pol δ might have important consequences both for lagging strand DNA
synthesis and for long patch BER. In this work, we addressed this issue by
constructing a series of synthetic gapped DNA templates with a single AP site
at different positions with respect to the downstream primer to be displaced
by DNA pol δ (see Fig.
1A). We show that an AP site immediately upstream of a
single- to double-strand DNA junction constitutes a strong block to the strand
displacement activity of DNA pol δ, even in the presence of RP-A and
PCNA. Such a block could be resolved only through a “polymerase
switch” involving the concerted physical and functional interaction of
DNA pol β and Fen-1. The closely related DNA pol λ could only
partially substitute for DNA pol β. Based on our data, we propose that
stalling of a replication fork by an AP site not only is a consequence of its
ability to inhibit nucleotide incorporation by the replicative DNA pols but
can also stem from its effects on strand displacement during Okazaki fragment
maturation. In summary, our data suggest the existence of a novel repair
pathway that might be important in preventing replication fork stalling and
identify a previously unnoticed deleterious effect of the AP site lesion on
normal cell metabolism.Open in a separate windowFIGURE 1.An abasic site immediately upstream of a double-stranded DNA region
inhibits the strand displacement activity of DNA polymerase δ. The
reactions were performed as described under “Experimental
Procedures.” A, schematic representation of the various DNA
templates used. The size of the resulting gaps is indicated in nt. The
position of the AP site on the 100-mer template strand is indicated relative
to the 3′ end. Base pairs in the vicinity of the lesion are indicated by
dashes. The size of the gaps (35–38 nt) is consistent with the
size of ssDNA covered by a single RP-A molecule, which has to be released
during Okazaki fragment synthesis when the DNA pol is approaching the
5′-end of the downstream fragment. When the AP site is covered by the
downstream terminator oligonucleotide (Gap-3 and Gap-1 templates) the
nucleotide placed on the opposite strand is C to mimic the situation generated
by spontaneous loss of a guanine or excision of an oxidized guanine, whereas
when the AP site is covered by the primer (nicked AP template), the nucleotide
placed on the opposite strand is A to mimic the most frequent incorporation
event occurring opposite an AP site. B, human PCNA was titrated in
the presence of 15 nm (lanes 2–4 and
10–12) or 30 nm (lanes 6–8 and
14–16) recombinant human four subunit DNA pol δ, on a
linear control (lanes 1–8) or a 38-nt gap control (lanes
9–16) template. Lanes 1, 5, 9, and 13, control
reactions in the absence of PCNA. C, human PCNA was titrated in the
presence of 60 nm DNA pol δ, on a linear AP (lanes
2–4) or 38-nt gap AP (lanes 6–9) template. Lanes
1 and 5, control reactions in the absence of PCNA. 相似文献
10.
《Inorganica chimica acta》1986,123(2):87-90
Derivatives of Co(II), Ni(II), Cu(II) and Zn(II) with 3′AMP and 2′AMP were synthesized and characterized by IR UV-Vis and fluorescence spectroscopy. There seems to be bonding of the metal ion to the base in all cases. The activation test, using the complexes as allosteric labels, was carried out with rabbit muscle glycogen phosphorylase b, but the enzyme was not activated, confirming that the phosphate group must necessarily be bonded to position 5′ of the ribose in order to activate this enzyme. 相似文献
11.
This study investigated the influence of the nature of oligonucleotides on the abilities to form antiparallel and parallel duplexes. Base pairing of homopurine DNA, 2’-O-MeRNA and RNA oligonucleotides with respective homopyrimidine DNA, 2’-O-MeRNA and RNA as well as chimeric oligonucleotides containing LNA resulted in the formation of 18 various duplexes. UV melting, circular dichroism and fluorescence studies revealed the influence of nucleotide composition on duplex structure and thermal stability depending on the buffer pH value. Most duplexes simultaneously adopted both orientations. However, at pH 5.0, parallel duplexes were more favorable. Moreover, the presence of LNA nucleotides within a homopyrimidine strand favored the formation of parallel duplexes. 相似文献
12.
13.
A key set of reactions for the initiation of new DNA strands during herpes
simplex virus-1 replication consists of the primase-catalyzed synthesis of
short RNA primers followed by polymerase-catalyzed DNA synthesis
(i.e. primase-coupled polymerase activity). Herpes primase
(UL5-UL52-UL8) synthesizes products from 2 to ∼13 nucleotides long.
However, the herpes polymerase (UL30 or UL30-UL42) only elongates those at
least 8 nucleotides long. Surprisingly, coupled activity was remarkably
inefficient, even considering only those primers at least 8 nucleotides long,
and herpes polymerase typically elongated <2% of the primase-synthesized
primers. Of those primers elongated, only 4–26% of the primers were
passed directly from the primase to the polymerase (UL30-UL42) without
dissociating into solution. Comparing RNA primer-templates and DNA
primer-templates of identical sequence showed that herpes polymerase greatly
preferred to elongate the DNA primer by 650–26,000-fold, thus accounting
for the extremely low efficiency with which herpes polymerase elongated
primase-synthesized primers. Curiously, one of the DNA polymerases of the host
cell, polymerase α (p70-p180 or p49-p58-p70-p180 complex), extended
herpes primase-synthesized RNA primers much more efficiently than the viral
polymerase, raising the possibility that the viral polymerase may not be the
only one involved in herpes DNA replication.Herpes simplex virus 1
(HSV-1)2 encodes seven
proteins essential for replicating its double-stranded DNA genome; five of
these encode the heterotrimeric helicase-primase (UL5-UL52-UL8 gene products)
and the heterodimeric polymerase (UL30-UL42 gene products)
(1,
2). The helicase-primase
unwinds the DNA at the replication fork and generates single-stranded DNA for
both leading and lagging strand synthesis. Primase synthesizes short RNA
primers on the lagging strand that the polymerase presumably elongates using
dNTPs (i.e. primase-coupled polymerase activity). These two protein
complexes are thought to replicate the viral genome on both the leading and
lagging strands (1,
2).Previous studies have focused on the helicase-primase and polymerase
separately. The helicase-primase contains three subunits, UL5, UL52, and UL8
in a 1:1:1 ratio
(3–5).
The UL5 subunit has helicase-like motifs and the UL52 subunit has primase-like
motifs, yet the minimal active complex that demonstrates either helicase or
primase activities contains both UL5 and UL52
(6,
7). Although the UL8 subunit
has no known catalytic activity, several functions have been proposed,
including enhancing helicase and primase activities, enhancing primer
synthesis on ICP8 (the HSV-1 single-stranded binding protein)-coated DNA
strands, and facilitating formation of the replisome
(8–12).
Although primase will synthesize short
(2–3
nucleotides long) primers on a variety of template sequences, synthesis of
longer primers up to 13 nucleotides long requires the template sequence,
3′-deoxyguanidine-pyrimidine-pyrimidine-5′
(13). Primase initiates
synthesis at the first pyrimidine via the polymerization of two purine NTPs
(13). Even after initiation at
this sequence, however, the vast majority of products are only 2–3
nucleotides long (13,
14).The herpes polymerase consists of the UL30 subunit, which has polymerase
and 3′ → 5′ exonuclease activities
(1,
2), and the UL42 subunit, which
serves as a processivity factor
(15–17).
Unlike most processivity factors that encircle the DNA, the UL42 protein binds
double-stranded DNA and thus directly tethers the polymerase to the DNA
(18). Using pre-existing DNA
primer-templates as the substrate, the heterodimeric polymerase (UL30-UL42)
incorporates dNTPs at a rate of 150 s–1, a rate much faster
than primer synthesis (for primers >7 nucleotides long, 0.0002–0.01
s–1) (19,
20).We examined primase-coupled polymerase activity by the herpes primase and
polymerase complexes. Although herpes primase synthesizes RNA primers
2–13 nucleotides long, the polymerase only effectively elongates those
at least 8 nucleotides long. Surprisingly, the polymerase elongated only a
small fraction of the primase-synthesized primers (<1–2%), likely
because of the polymerase elongating RNA primer-templates much less
efficiently than DNA primer-templates. In contrast, human DNA polymerase
α (pol α) elongated the herpes primase-synthesized primers very
efficiently. The biological significance of these data is discussed. 相似文献
14.
The genus Planinasus Cresson is revised and includes 18 extant and one fossil species. We clarify the status of the three previously described species and describe 15 new species as follows (type locality in parenthesis): Planinasus aenigmaticus (Colombia. Bogota: Bogota (04°35.8''N, 74°08.8''W)), Planinasus neotropicus (Panama. Canal Zone: Barro Colorado Island (09°09.1''N, 79°50.8''W)), Planinasus kotrbae (Ecuador. Orellana: Rio Tiputini Biodiversity Station (0°38.2''S, 76°08.9''W)), Planinasus miradorus (Brazil. Maranhão: Parque Estadual Mirador, Base da Geraldina (06°22.2''S, 44°21.8''W)), Planinasus tobagoensis (Trinidad and Tobago. Tobago. St. John: Parlatuvier (11°17.9''N, 60°39''W)), Planinasus xanthops (Ecuador. Orellana: Rio Tiputini Biodiversity Station (0°38.2''S, 76°8.9''W)), Planinasus argentifacies (Peru. Madre de Dios: Río Manu, Pakitza (11°56.6''S, 71°16.9''W; 250 m)), Planinasus insulanus (Dominican Republic. La Vega: near Jarabacoa, Salto Guasara (19°04.4''N, 70°42.1''W, 680 m)), Planinasus nigritarsus (Guyana. Conservation of Ecological Interactions and Biotic Associations (CEIBA; ca. 40 km S Georgetown; 06°29.9''N, 58°13.1''W)), Planinasus atriclypeus (Brazil. Rio de Janeiro: Rio de Janeiro, Floresta da Tijuca (22°57.6''S, 43°16.4''W)), Planinasus atrifrons (Bolivia. Santa Cruz: Ichilo, Buena Vista (4-6 km SSE; Hotel Flora y Fauna; 17°29.95''S, 63°33.15''W; 4-500 m)), P. flavicoxalis (West Indies. Dominica. St. David: 1.6 km N of junction of roads to Rosalie and Castle Bruce (15°23.8''N, 61°18.6''W)), Planinasus mcalpineorum (Mexico. Chiapas: Cacahoatan (7 km N; 15°04.1''N, 92°07.4''W)), Planinasus nigrifacies (Brazil. São Paulo: Mogi das Cruzes, Serra do Itapeti (23°31.5''S, 46°11.2''W)), Planinasus obscuripennis (Peru. Madre de Dios: Río Manu, Erika (near Salvación; 12°50.7''S, 71°23.3''W; 550 m)). In addition to external characters, we also describe and illustrate structures of the male terminalia and for Planinasus kotrbae
sp. n., the internal female reproductive organs. Detailed locality data and distribution maps for all species are provided. For perspective and to facilitate genus-group and species-group recognition, the family Periscelididae and subfamily Stenomicrinae are diagnosed and for the latter, a key to included genera is provided. 相似文献
15.
Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression. 相似文献
16.
cDNA
corresponding to the GA4 gene of
Arabidopsis thaliana L. (Heynh.) was
expressed in Escherichia coli, from which cell lysates
converted [14C]gibberellin (GA)9 and
[14C]GA20 to radiolabeled GA4 and
GA1, respectively, thereby confirming that
GA4 encodes a GA 3β-hydroxylase. GA9 was
the preferred substrate, with a Michaelis value of 1 μm
compared with 15 μm for GA20. Hydroxylation
of these GAs was regiospecific, with no indication of
2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant
enzyme to hydroxylate a range of other GA substrates was investigated.
In general, the preferred substrates contained a polar bridge between
C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated
analogs. Therefore, no activity was detected using
GA12-aldehyde, GA12, GA19,
GA25, GA53, or GA44 as the open
lactone (20-hydroxy-GA53), whereas GA15,
GA24, and GA44 were hydroxylated to
GA37, GA36, and GA38, respectively.
The open lactone of GA15 (20-hydroxy-GA12) was
hydroxylated but less efficiently than GA15. In contrast to
the free acid, GA25 19,20-anhydride was 3β-hydroxylated
to give GA13. 2,3-Didehydro-GA9 and
GA5 were converted by recombinant GA4 to the corresponding
epoxides 2,3-oxido-GA9 and GA6.Dwarf mutants with reduced biosynthesis of the GA plant hormones
have been valuable tools in studies of the function of these compounds
(Ross, 1994). In Arabidopsis thaliana, mutations
at six loci (GA1-GA6) that result in reduced GA
biosynthesis have been identified (Koorneef and van der Veen, 1980;
Sponsel et al., 1997), and three of these loci have recently been
cloned. The GA1 locus was isolated by genomic subtraction
(Sun et al., 1992) and shown by heterologous expression in
Escherichia coli to encode the enzyme that cyclizes
geranylgeranyl diphosphate to copalyl diphosphate (Sun and Kamiya,
1994). This enzyme was formerly referred to as ent-kaurene
synthase A but has been renamed copalyl diphosphate synthase
(Hedden and Kamiya, 1997; MacMillan, 1997). The GA5
locus was shown to correspond to one of the GA 20-oxidase genes (Xu et
al., 1995), the products of which catalyze the conversion of
GA12 to GA9 and
GA53 to GA20 (Phillips et
al., 1995; Xu et al., 1995). GA 20-oxidases are
2-oxoglutarate-dependent dioxygenases that are encoded by small
multigene families, members of which are differentially expressed in
plant tissues (Phillips et al., 1995; Garcia-Martinez et al., 1997).The GA4 locus was isolated by T-DNA tagging and, on the
basis of the derived amino acid sequence, was also shown to encode a
dioxygenase (Chiang et al., 1995). Several lines of evidence indicate
that the GA4 gene encodes a GA 3β-hydroxylase. Shoots of a
ga4 mutant, all alleles of which are semidwarf, contained
reduced concentrations of the 3β-hydroxy GAs
GA1, GA4, and
GA8 compared with the Landsberg erecta
wild type, whereas levels of immediate precursors to these GAs were
elevated (Talon et al., 1990). Furthermore, metabolism of
[13C]GA20 to
[13C]GA1 was
substantially less in the mutant than in the wild type (Kobayashi et
al., 1994). In the present paper we confirm by functional expression of
its cDNA in E. coli that GA4 encodes a GA
3β-hydroxylase. In addition, we determine the substrate specificity
of recombinant GA4 using a number of C20- and
C19-GAs and show by kinetic analysis that the enzyme
has a higher affinity for GA9 than for
GA20, which is consistent with the
non-13-hydroxylation pathway predominating in Arabidopsis (Talon et
al., 1990). 相似文献
17.
The tiger beetle fauna of the Balkan Peninsula is one of the richest in Europe and includes 19 species or 41% of the European tiger beetle fauna. Assembled by their biogeographical origins, the Balkan tiger beetle species fall into 14 different groups that include, Mediterranean, Middle Oriental, Central Asiatic, Euro-Siberian, South and East European, Pannonian-Sarmatian, West Palaearctic, Turano-European and Afrotropico Indo-Mediterranean species. The Mediterranean Sclerophyl and the Pontian Steppe are the Balkan biogeographical provinces with the highest species richness, while the Balkan Highlands has the lowest Cicindelidae diversity. Most species are restricted to single habitat types in lowland areas of the Balkan Peninsula and only Calomera aulica aulica and Calomera littoralis nemoralis occur in respectively 3 and 4 different types of habitat. About 60% of all Balkan Cicindelidae species are found in habitats potentially endangered by human activity. 相似文献
18.
Probing the Conformation of the Fibronectin III1�C2
Domain by Fluorescence Resonance Energy
Transfer
Nancy W. Karuri Zong Lin Hays S. Rye Jean E. Schwarzbauer 《The Journal of biological chemistry》2009,284(6):3445-3452
Fibronectin (FN) matrix is crucial for cell and tissue functions during
embryonic development, wound healing, and oncogenesis. Assembly of FN matrix
fibrils requires FN domains that mediate interactions with integrin receptors
and with other FN molecules. In addition, regulation of FN matrix assembly
depends on the first two FN type III modules, III1 and
III2, which harbor FN-binding sites. We propose that interactions
between these two modules sequester FN-binding sites in soluble FN and that
these sites become exposed by FN conformational changes during assembly. To
test the idea that III1–2 has a compact conformation, we
constructed CIIIY, a conformational sensor of III1–2 based on
fluorescent resonance energy transfer between cyan and yellow fluorescent
proteins conjugated at its N and C termini. We demonstrate energy transfer in
CIIIY and show that fluorescent resonance energy transfer was eliminated by
proteolysis and by treatment with mild denaturants that disrupted
intramolecular interactions between the two modules. We also show that
mutations of key charged residues resulted in conformational changes that
exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively,
these results support a conformation-dependent mechanism for the regulation of
FN matrix assembly by III1–2.Fibronectin (FN)3
is a 500-kDa modular dimeric protein and a major component of the
extracellular matrix. It exists in the blood and other body fluids as a
soluble compact molecule and undergoes cell-mediated assembly to form an
insoluble three-dimensional fibrillar matrix (reviewed in Ref.
1). The process of FN matrix
assembly has been implicated in embryonic development, wound healing, and
cancer
(2–4).
FN is composed of type I–III modules, and sets of these modules comprise
binding domains for cells and for other extracellular matrix components (see
Fig. 1A). Three of
these binding domains are essential for matrix assembly
(1). Integrin receptor
interactions with the cell-binding domain tether disulfide-bonded FN dimers to
the cell surface, where FN-FN interactions involving the N-terminal assembly
domain form dimers into fibrils. In addition to these essential domains, other
FN-binding sites have been implicated in assembly. In particular, the
III1–2 FN-binding domain plays a regulatory role in matrix
assembly. Within this domain reside a cryptic FN-binding site in
III1 and a site available for FN binding in the native form of
III2
(5–8).
Recombinant FN lacking III1 is assembled into a matrix at wild-type
levels, but that lacking the III1–2 domain results in short
immature FN fibrils (8).
Peptides derived from the III1–2 domain or antibodies against
III1–2 block matrix assembly by cultured cells
(9–11).
Furthermore, FN binding to this region is enhanced when FN is mechanically
stretched (12). Taken
together, these results suggest that conformational changes in the
III1–2 domain may control its interactions during FN
assembly.Open in a separate windowFIGURE 1.The FN III1–2 FRET conformational sensor.
A, representation of the domain structure of FN and major interaction
sites. FN is composed of repeating modules that form binding domains for other
FN molecules, cell receptors, and other extracellular matrix components as
indicated. The first two type III modules III1 and III2
(black), have FN-binding sites and regulate FN matrix assembly. The
N-terminal 70-kDa region contains a matrix assembly domain with FN-binding
activity. The cell-binding domain (cell), the heparin-binding domain
(heparin), the dimerization site (SS), and the alternatively
spliced type IIIA (A), IIIB (B), and variable regions
(V) are indicated. 70kD, N-terminal 70-kDa FN fragment.
B, schematic of proposed model of III1–2 domain
conformation. Panel i, in solution, the FN-binding sites in
III1 and III2 (hatched areas) are sequestered
through domain orientations that are facilitated by the linker between modules
(thin line). Panel ii, binding sites are exposed through
conformational changes resulting from cell-mediated extension of FN
(arrows). The length of the linker and the height and width of the
modules are drawn to scale for a linear peptide and published data on FN type
III modules, respectively. C, ribbon diagram representation of CIIIY,
a FRET sensor of the model in B (panel i), oriented with N
and C termini 50 Å apart. CIIIY consists of the III1–2
domain with CFP at the N terminus and YFP at the C terminus.To more fully understand the roles of native and cryptic FN-binding sites
in matrix assembly, the conformational dynamics of III1–2
must be characterized. One approach to this problem is to tag
III1–2 with fluorescent probes, which, in conjunction with
fluorescent resonance energy transfer (FRET), create a molecular
conformational sensor. FRET involves the radiationless transfer of energy from
an excited donor fluorophore to an acceptor fluorophore, a process that is
very sensitive to the distance between the two fluorophores
(13–15).
Two fluorescent protein variants, cyan fluorescent protein (CFP) and yellow
fluorescent protein (YFP), are highly related to green fluorescent protein
(GFP). Because the emission spectrum of CFP is well matched to the excitation
spectrum of YFP, these two fluorophores have been widely used as a
donor-acceptor pair in FRET studies
(13–15).In this study, we describe a FRET conformational sensor designed to test
the idea that intramolecular interactions between III1 and
III2 sequester key FN-binding and assembly sites. We show that
III1–2 with CFP and YFP fused to the N and C termini,
respectively, displays a clear FRET signal, indicating that the attached
fluorescent proteins and thus the ends of III1–2 are in close
proximity. FRET data from III1–2 mutants support the presence
of a stabilizing intermodule salt bridge that regulates FN-binding
activity. 相似文献
19.
A new ligand N-Nicotinoyl-N-o-hydroxythiobenzhydrazide (H2Notbh) forms complexes [Mn(Notbh)(H2O)], [M(Notbh)] [M=Ni(II) Cu(II) and Zn(II)] which were characterized by various physico-chemical techniques. All the metal complexes were observed to inhibit the growth of tumor in vitro, whereas, ligand did not. In vivo administration of these complexes resulted in prolongation of survival of tumor bearing mice. Tumor bearing mice administered with metal complexes showed reversal of tumor growth associated induction of apoptosis in lymphocytes. The paper discusses the possible mechanisms and therapeutic implication of the H2Notbh and its metal complexes in tumor regression and tumor growth associated immunosuppression. 相似文献
20.
Antisense strategies that target DNA·RNA hybrid structures offer potential for the development of new therapeutic drugs. The α-sarcin loop region of the 16S rRNA domain has been shown to be a high value target for such strategies. Herein, aminoglycoside interaction with three RNA·DNA α-sarcin targeted duplexes (rR·dY, rR·S-dY, and rR·2'OMe-rY) have been investigated to determine the overall effect of aminoglycoside interaction on the stability, affinity, and conformation of these hybrid duplexes. To this end, UV thermal denaturation, circular dichroism spectroscopy, fluorescence intercalator displacement, and ITC as well as DSC calorimetry experiments were carried out. The results suggest the following. (1) Of all the aminoglycosides studied, neomycin confers the highest thermal stability on all three hybrid duplexes studied. (2) There is no appreciable difference in aminoglycoside-induced thermal stability between the unmodified rR·dY and phophorothioate modified rR·S-dY duplexes. (3) The rR·2'OMe-rY duplexes thermal stability is slightly less than the other two hybrids. (4) In all three duplexes, aminoglycoside-induced thermal stability decreased as the number of amino groups decreased. (5) CD scans revealed similar spectra for the rR·dY and rR·S-dY duplexes as well as a more pronounced A-form signal for the rR·2'OMe-rY duplex. (6) FID assays paralleled the CD results, yielding similar affinity values between the rR·dY and rR·S-dY duplexes and higher affinities with the rR·2'OMe-rY duplex. (7) The overall affinity trend between aminoglycosides and the three duplexes was determined to be neomycin > paromomycin > neamine > ribostamycin. (8) ITC K(a) values revealed similar binding constants for the rR·dY and rR·S-dY duplexes with rR·dY having a K(1) of (1.03 ± 0.58) × 10(7) M(-1) and K(2) of (1.13 ± 0.07) × 10(5) M(-1) while rR·S-dY produced a K(1) of (1.17 ± 0.54) × 10(7) M(-1) and K(2) of (1.27 ± 0.69) × 10(5) M(-1). (8) The rR·2'OMe-rY produced a slightly higher binding constant values with a K(1) of (1.25 ± 0.24) × 10(7) M(-1) and K(2) of (3.62 ± 0.18) × 10(5) M(-1). (9) The ΔT(m)-derived K(Tm) of 3.81 × 10(7) M(-1) for rR·S-dY was in relative agreement with the corresponding K(1) of 1.17 × 10(7) M(-1) derived constant from the fitted ITC. These results illustrate that the increased DNA·RNA hybrid duplex stability in the presence of aminoglycosides can help extend the roles of aminoglycosides in designing modified ODNs for targeting RNA. 相似文献