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81.
Grden M Podgorska M Kocbuch K Szutowicz A Pawelczyk T 《Archives of biochemistry and biophysics》2006,455(1):10-17
Adenosine among other factors is known to regulate the growth and function of cardiac fibroblasts (CFs). Its action is mediated by cell-surface receptors linked to a variety of signaling systems. The goal of present work was to examine the effects of glucose and insulin on adenosine receptors (ARs) mRNA and protein level in primary culture of rat CFs by means of real-time PCR and Western blot. Elevated glucose level increased the expression of A(1)-AR, A(2A)-AR, decreased the expression of A(3)-AR, and had no effect on A(2B)-AR expression. On the other hand insulin suppressed the expression of A(1)-AR, and A(2B)-AR, and had no effect on A(2A)-AR and A(3)-AR expression. Our measurements showed that accumulation of cAMP in response to ARs agonists correlated well with the changes in receptors expression level. These results indicate that changes in glucose and insulin level independently and differentially regulate the ARs expression and functional state in CFs. 相似文献
82.
Tibor Pastor Gabriele Talotti Marzena Anna Lewandowska Franco Pagani 《Nucleic acids research》2009,37(21):7258-7267
We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations. 相似文献
83.
Carla S. D’Angelo Marzena Gajecka Chong A. Kim Andrew J. Gentles Caron D. Glotzbach Lisa G. Shaffer Célia P. Koiffmann 《Human genetics》2009,125(5-6):551-563
The mechanisms involved in the formation of subtelomeric rearrangements are now beginning to be elucidated. Breakpoint sequencing analysis of 1p36 rearrangements has made important contributions to this line of inquiry. Despite the unique architecture of segmental duplications inherent to human subtelomeres, no common mechanism has been identified thus far and different nonexclusive recombination–repair mechanisms seem to predominate. In order to gain further insights into the mechanisms of chromosome breakage, repair, and stabilization mediating subtelomeric rearrangements in humans, we investigated the constitutional rearrangements of 1p36. Cloning of the breakpoint junctions in a complex rearrangement and three non-reciprocal translocations revealed similarities at the junctions, such as microhomology of up to three nucleotides, along with no significant sequence identity in close proximity to the breakpoint regions. All the breakpoints appeared to be unique and their occurrence was limited to non-repetitive, unique DNA sequences. Several recombination- or cleavage-associated motifs that may promote non-homologous recombination were observed in close proximity to the junctions. We conclude that NHEJ is likely the mechanism of DNA repair that generates these rearrangements. Additionally, two apparently pure terminal deletions were also investigated, and the refinement of the breakpoint regions identified two distinct genomic intervals ~25-kb apart, each containing a series of 1p36 specific segmental duplications with 90–98% identity. Segmental duplications can serve as substrates for ectopic homologous recombination or stimulate genomic rearrangements. 相似文献
84.
Rui Gong Bang K. Vu Yang Feng DaRue A. Prieto Marzena A. Dyba Joseph D. Walsh Ponraj Prabakaran Timothy D. Veenstra Sergey G. Tarasov Rieko Ishima Dimiter S. Dimitrov 《The Journal of biological chemistry》2009,284(21):14203-14210
The immunoglobulin (Ig) constant CH2 domain is critical for antibody
effector functions. Isolated CH2 domains are promising as scaffolds for
construction of libraries containing diverse binders that could also confer
some effector functions. However, previous work has shown that an isolated
murine CH2 domain is relatively unstable to thermally induced unfolding. To
explore unfolding mechanisms of isolated human CH2 and increase its stability
γ1 CH2 was cloned and a panel of cysteine mutants was constructed. Human
γ1 CH2 unfolded at a higher temperature (Tm = 54.1
°C, as measured by circular dichroism) than that previously reported for a
mouse CH2 (41 °C). One mutant (m01) was remarkably stable
(Tm = 73.8 °C). Similar results were obtained by
differential scanning calorimetry. This mutant was also significantly more
stable than the wild-type CH2 against urea induced unfolding (50% unfolding at
urea concentration of 6.8 m versus 4.2 m). The
m01 was highly soluble and monomeric. The existence of the second disulfide
bond in m01 and its correct position were demonstrated by mass spectrometry
and nuclear magnetic resonance spectroscopy, respectively. The loops were on
average more flexible than the framework in both CH2 and m01, and the overall
secondary structure was not affected by the additional disulfide bond. These
data suggest that a human CH2 domain is relatively stable to unfolding at
physiological temperature, and that both CH2 and the highly stable mutant m01
are promising new scaffolds for the development of therapeutics against human
diseases.Monoclonal antibodies
(mAbs)2 with high
affinity and specificity are now well established therapeutics and invaluable
tools for biological research. It appears that their use will continue to
expand in both targets and disease indications. However, a fundamental problem
for full-size mAbs that limits their applications is their poor penetration
into tissues (e.g. solid tumors) and poor or absent binding to
regions on the surface of some molecules (e.g. on the HIV envelope
glycoprotein) that are accessible by molecules of smaller size. Antibody
fragments, e.g. Fabs (∼60 kDa) or single chain Fv fragments
(scFvs) (20∼30 kDa), are significantly smaller than full-size antibodies
(∼150 kDa), and have been used as imaging reagents and candidate
therapeutics. Even smaller fragments of antibodies are of great interest and
advantageous for pharmaceutical applications including cancer targeting and
imaging.During the last decade a large amount of work has been aimed at developing
of small size binders with scaffolds based on various highly stable human and
non-human molecules
(1–8).
A promising direction is the development of binders based on the heavy or
light chain variable region of an antibody; these fragments ranging in size
from 11 kDa to 15 kDa were called “domain antibodies” or
“dAbs” (7,
9). A unique kind of antibodies
composed only of heavy chains are naturally formed in camels, dromedaries, and
llamas, and their variable regions can also recognize antigens as single
domain fragments (10). Not
only is the overall size of the dAbs much smaller than that of full-size
antibodies but also their paratopes are concentrated over a smaller area so
that the dAbs provide the capability of interacting with novel epitopes that
are inaccessible to conventional antibodies or antibody fragments with paired
light and heavy chain variable domains.The structure of the constant antibody domains is similar to that of the
variable domains consisting of β-strands connected mostly with loops or
short helices. The second domain of the α, δ, and γ heavy
chain constant regions, CH2, is unique in that it exhibits very weak
carbohydrate-mediated interchain protein-protein interactions in contrast to
the extensive interchain interactions that occur between the other domains.
The expression of murine CH2 in bacteria, which does not support
glycosylation, results in a monomeric domain
(11). It has been hypothesized
that the CH2 domain (CH2 of IgG, IgA, and IgD, and CH3 of IgE and IgM) could
be used as a scaffold and could offer additional advantages compared with
those of dAbs because it contains binding sites or portions of binding sites
conferring effector and stability functions
(12).It was found previously that an isolated murine CH2 is relatively unstable
at physiological temperature with a temperature of 50% unfolding
(Tm) slightly higher than 37 °C
(11). We have hypothesized
that human CH2 would exhibit different stability because of significant
differences in the sequence compared with its murine counterpart. Therefore,
we have extensively characterized the stability of an isolated unglycosylated
single CH2 domain. We found that its stability is significantly higher than
the previously reported for the murine CH2. We further increased the stability
of the human CH2 by engineering an additional disulfide bond between the A and
G strands. One of the newly developed mutants, denoted as m01, exhibited
significantly higher stability (Tm = 73.8 °C) than
that of wild type CH2. We suggest that both the wild type CH2 and the newly
developed mutant, m01, could be used as scaffolds for binders. These results
also demonstrate for the first time that the stability of constant antibody
domains can be dramatically increased by engineering of an additional
disulfide bond. The increase in stability of isolated domains may result in an
increase in stability of larger antibody fragments, e.g. Fc, and
therefore could have implications as a general method for increasing antibody
stability. Thus, it appears that further development of CH2 and its more
stable variants as scaffolds could provide new opportunities for
identification of potentially useful therapeutics. 相似文献
85.
86.
Monika Sakowicz‐Burkiewicz Katarzyna Kocbuch Marzena Grden Andrzej Szutowicz Tadeusz Pawelczyk 《Journal of cellular biochemistry》2010,109(2):396-405
Development of diabetes is associated with altered expression of adenosine receptors (ARs). Some of these alterations might be attributed to changes in insulin concentration. This study was undertaken to investigate the possible insulin effect on ARs level, and to determine the signaling pathway utilized by insulin to regulate the expression of ARs in rat B lymphocytes. Western blot analysis of B lymphocytes protein extracts indicated that all four ARs were present at detectable levels in the cells cultured for 24 h without insulin (≤10?11 M), although the protein band of A2A‐AR was barely visible. Inclusion of insulin (10?8 M) in the culture medium resulted in an increase of A1‐AR and A2A‐AR protein levels and a significant decrease of A2B‐AR protein, whereas the protein level of A3‐AR remained unchanged. Alterations in the ARs protein content were accompanied by changes in the ARs mRNA levels. Increase of the insulin concentration from 10?11 to 10?8 M resulted in 50% decrease of A2B‐AR mRNA level and two‐, and threefold increase of A1‐AR and A2A‐AR mRNA levels, respectively. Pretreatment of B cells with cycloheximide completely blocked the insulin action on A1‐AR and A2A‐AR mRNA, but not on A2B‐AR expression. Detailed pharmacological analysis demonstrated that insulin‐induced A1‐AR and A2A‐AR mRNA expression through the Ras/Raf‐1/MEK/ERK pathway. The insulin effect on A2B‐AR expression was blocked by p38 MAP kinase inhibitor (SB 203580). Concluding, elevated insulin concentration differentially affects the expression of ARs in B lymphocytes in a fashion that might enhance the various immunomodulatory effects of adenosine. J. Cell. Biochem. 109: 396–405, 2010. © 2009 Wiley‐Liss, Inc. 相似文献
87.
Renata Grąbkowska Aleksandra Królicka Wojciech Mielicki Marzena Wielanek Halina Wysokińska 《Acta Physiologiae Plantarum》2010,32(4):665-673
A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types
of explants and acetosyringone (AS) (200 μM). The highest frequency of transformation (over 50% of explants forming roots
at the infected sites after 6 weeks of culture on Lloyd and McCown (WP) medium) was achieved using a combination of nodal
stem explants and A. rhizogenes strain A4. The addition of 200 μM AS to root induction medium was found to enhance hairy root induction but its effect varied
depending on bacterial strain and explant type. Three of the most vigorously growing hairy root clones of H. procumbens were chosen and analyzed for accumulation of iridoid and phenylethanoid glycosides. The transgenic nature of these root clones
was confirmed by PCR amplification; they were positive for rolB and rolC genes. Harpagoside, verbascoside and isoverbascoside were identified by HPLC and LC–ESI-MS as the major compounds from all
analyzed hairy root clones. The Hp-3 root clone showed the higher harpagoside content (0.32 mg g−1 dry wt.) compared with other analyzed transformed and non-tuberized untransformed roots of H. procumbens. However, the level of the compound in the hairy root clone was lower than that detected in a sample of commercially available
root tubers of H. procumbens. The Hp-3 root clone also produced high amounts of verbascoside and isoverbascoside (8.12 mg g−1 dry wt. and 9.97 mg g−1 dry wt., respectively) comparable to those found in root tubers. 相似文献
88.
Ulrike Muus Curtis Hose Wei Yao Teresa Kosakowska-Cholody David Farnsworth Marzena Dyba George T. Lountos David S. Waugh Anne Monks Terrence R. Burke Christopher J. Michejda 《Bioorganic & medicinal chemistry》2010,18(12):4535-4541
The current paper presents the synthesis and evaluation of a series of maleimides that were designed to inhibit the Cdc25 phosphatase by alkylation of catalytically essential cysteine residues. Although in HepB3 cell culture assays the analogues did exhibit antiproliferative IC50 values ranging from sub-micromolar to greater than 100 μM, inhibition of Cdc25 through cysteine alkylation could not be demonstrated. It was also found that analysis using fluorescence activated cell sorting (FACS) following treatment with the most potent analogue (1t) did not provide data consistent with inhibition at one specific point in the cell cycle, as would be expected if Cdc25A were inhibited. Further studies with a subset of analogues resulted in a correlation of antiproliferative potencies with activation of the unfolded protein response (UPR). The UPR is a regulatory pathway that temporarily suspends protein production when misfolding of proteins occurs within the endoplastic reticulum (ER). In addition, ER chaperones that promote proper refolding become up-regulated. If cellular damage cannot be resolved by these mechanisms, then the UPR can initiate apoptosis. The current study indicates that these maleimide analogues lead to UPR activation, which is predictive of the selective antiproliferative activity of the series. 相似文献
89.
Chong Li Marzena Pazgier Jing Li Changqing Li Min Liu Guozhang Zou Zhenyu Li Jiandong Chen Sergey G. Tarasov Wei-Yue Lu Wuyuan Lu 《The Journal of biological chemistry》2010,285(25):19572-19581
A retro-inverso peptide is made up of d-amino acids in a reversed sequence and, when extended, assumes a side chain topology similar to that of its parent molecule but with inverted amide peptide bonds. Despite their limited success as antigenic mimicry, retro-inverso isomers generally fail to emulate the protein-binding activities of their parent peptides of an α-helical nature. In studying the interaction between the tumor suppressor protein p53 and its negative regulator MDM2, Sakurai et al. (Sakurai, K., Chung, H. S., and Kahne, D. (2004) J. Am. Chem. Soc. 126, 16288–16289) made a surprising finding that the retro-inverso isomer of p53(15–29) retained the same binding activity as the wild type peptide as determined by inhibition enzyme-linked immunosorbent assay. The authors attributed the unusual outcome to the ability of the d-peptide to adopt a right-handed helical conformation upon MDM2 binding. Using a battery of biochemical and biophysical tools, we found that retro-inverso isomerization diminished p53 (15,–29) binding to MDM2 or MDMX by 3.2–3.3 kcal/mol. Similar results were replicated with the C-terminal domain of HIV-1 capsid protein (3.0 kcal/mol) and the Src homology 3 domain of Abl tyrosine kinase (3.4 kcal/mol). CD and NMR spectroscopic as well as x-ray crystallographic studies showed that d-peptide ligands of MDM2 invariably adopted left-handed helical conformations in both free and bound states. Our findings reinforce that the retro-inverso strategy works poorly in molecular mimicry of biologically active helical peptides, due to inherent differences at the secondary and tertiary structure levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structure level. 相似文献
90.
Rathaur S Fischer P Domagalski M Walter RD Liebau E 《Experimental parasitology》2003,103(3-4):177-181
The nucleotide sequences for Brugia malayi and Wuchereria bancrofti GST have been submitted to EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under Accession Nos. Y12788 and AY195867. 相似文献