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101.
Mature and dried seeds from three species of the Acer genus, which differed in desiccation tolerance, were analyzed. The three species investigated were as follows: Acer platanoides L. (Norway maple, orthodox, A1 and A2 seedlots); Acer pseudoplatanus L. (sycamore, recalcitrant, B1 and B2 seedlots); and Acer saccharinum (silver maple, recalcitrant, C1 and C2 seedlots). We compared the appearance of dehydrins and small heat shock proteins in seedlots originating from cropping years that differed in weather conditions, which were monitored in detail during seed development. The experiments showed that three main dehydrins with approximate molecular weights of 46, 35, and 23?kDa were characteristic of all examined Acer species seeds. The three proteins were present in the A1 and A2 seedlots of the orthodox category Norway maple seeds and were noted either individually or together in the B1, B2, C1, and C2 seedlots of recalcitrant category seeds. It was found that one major small heat shock protein existed with a molecular mass of 22?kDa and was detectable at high concentrations in all seeds of the studied Acer species; after the seeds were dried, the content of this protein significantly increased. The potential modulation of dehydrin expression by environmental factors such as developmental heat sum and rainfall is discussed in the present work. The influence of water removal, which is caused by seed drying, in seeds of the same genus and belonging to the orthodox and recalcitrant categories is also explored. 相似文献
102.
Nowakowska E Schulz T Molenda N Schillers H Prehm P 《Journal of cellular biochemistry》2012,113(1):156-164
We recently discovered that hyaluronan was exported from fibroblasts by MRP5 and from epithelial cells by cystic fibrosis (CF) transmembrane conductance regulator (CFTR) that was known as a chloride channel. On this basis we developed membrane permeable analogs of hyaluronan disaccharide as new class of compounds to modify their efflux. We found substances that activated hyaluronan export from human breast cancer cells. The most active compound 2-(2-acetamido-3,5-dihydroxyphenoxy)-5-aminobenzoic acid (Hylout4) was tested for its influence on the activity of epithelial cells. It activated the ion efflux by normal and defective ΔF508-CFTR. It also enhanced the plasma membrane concentration of the ΔF508-CFTR protein and reduced the transepithelial resistance of epithelial cells. In human trials of healthy persons, it caused an opening of CFTR in the nasal epithelium. Thus compound Hylout4 is a corrector that recovered ΔF508-CFTR from intracellular degradation and activated its export function. 相似文献
103.
Gajecka M Pavlicek A Glotzbach CD Ballif BC Jarmuz M Jurka J Shaffer LG 《Human genetics》2006,120(4):519-526
Although approximately 1 in 500 individuals carries a reciprocal translocation, little is known about the mechanisms that result in their formation. We analyzed the sequences surrounding the breakpoints in three unbalanced translocations of 1p and 9q, all of which were designated t(1;9)(p36.3;q34), to investigate the presence of sequence motifs that might mediate nonhomologous end joining (NHEJ). The breakpoint regions were unique in all individuals. Two of three translocations demonstrated insertions and duplications at the junctions, suggesting NHEJ in the formation of the rearrangements. No homology was identified in the breakpoint regions, further supporting NHEJ. We found translin motifs at the breakpoint junctions, suggesting the involvement of translin in the joining of the broken chromosome ends. We propose a model for balanced translocation formation in humans similar to transposition in bacteria, in which staggered nicks are repaired resulting in duplications and insertions at the translocation breakpoints. 相似文献
104.
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
105.
Aneta Monika Białkowska Hubert Cieśliński Karolina Maria Nowakowska Józef Kur Marianna Turkiewicz 《Archives of microbiology》2009,191(11):825-835
A psychrotrophic bacterium producing a cold-adapted β-galactosidase upon growth at low temperatures was classified as Arthrobacter sp. 20B. A genomic DNA library of strain 20B introduced into Escherichia coli TOP10F′ and screening on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)-containing agar plates led to the isolation of β-galactosidase gene. The β-galactosidase gene (bgaS) encoding a protein of 1,053 amino acids, with a calculated molecular mass of 113,695 kDa. Analysis of the amino acid sequence
of BgaS protein, deduced from the bgaS ORF, suggested that it is a member of the glycosyl hydrolase family 2. A native cold-adapted β-galactosidase was purified
to homogeneity and characterized. It is a homotetrameric enzyme, each subunit being approximately 116 kDa polypeptide as deduced
from native and SDS–PAGE, respectively. The β-galactosidase was optimally active at pH 6.0–8.0 and 25°C. P-nitrophenyl-β-d-galactopyranoside (PNPG) is its preferred substrate (three times higher activity than for ONPG—o-nitrophenyl-β-d-galactopyranoside). The Arthrobacter sp. 20B β-galactosidase is activated by thiol compounds (53% rise in activity in the presence of 10 mM 2-mercaptoethanol),
some metal ions (activity increased by 50% for Na+, K+ and by 11% for Mn2+) and inactivated by pCMB (4-chloro-mercuribenzoic acid) and heavy metal ions (Pb2+, Zn2+, Cu2+). 相似文献
106.
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
107.
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
108.
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
109.
110.
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