Sulfated glycosaminoglycans (GAGs) are
linear polysaccharides of repeating disaccharide sequences on which are
superimposed highly complex and variable patterns of sulfation,
especially in heparan sulfate (HS). HS and the structurally related
heparin exert important biological functions, primarily by interacting
with proteins and regulating their activities. Evidence is accumulating
that these interactions depend on specific saccharide sequences, but
the lack of simple, direct techniques for sequencing GAG saccharides
has been a major obstacle to progress. We describe how HS and heparin
saccharides can be sequenced rapidly by using an integrated strategy
with chemical and enzymic steps. Attachment of a reducing-end
fluorescent tag establishes a reading frame. Partial selective chemical
cleavage at internal N-sulfoglucosamine residues with
nitrous acid then creates a set of fragments of defined sizes.
Subsequent digestion of these fragments with combinations of
exosulfatases and exoglycosidases permits the selective removal of
specific sulfates and monosaccharides from their nonreducing ends. PAGE
of the products yields a pattern of fluorescent bands from which the
saccharide sequence can be read directly. Data are presented on
sequencing of heparin tetrasaccharides and hexasaccharides of known
structure; these data show the accuracy and versatility of this
sequencing strategy. Data also are presented on the application of the
strategy to the sequencing of an HS decasaccharide of unknown
structure. Application and further development of this sequencing
Synthetic benzamide derivatives were investigated for their ability to inhibit histone deacetylase (HDA). In this study, one of the most active benzamide derivatives, MS-27-275, was examined with regard to its biological properties and antitumor efficacy. MS-27-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as sodium butyrate and trichostatin A; MS-27-275 induced p21WAF1/CIP1 and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-fluorouracil, and, of interest, the accumulation of p21WAF1/CIP1 tended to be faster and greater in the cell lines sensitive to MS-27-275. MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-fluorouracil. A structurally analogous compound to MS-27-275 without HDA-inhibiting activity showed neither the biological effects in cell culture nor the in vivo therapeutic efficacy. These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.
Site-specific recombination is responsible for a broad range of biological phenomena, including DNA inversion, resolution of transposition intermediates, and the integration and excision of bacteriophage genomes. Integration of mycobacteriophage L5 is catalyzed by a phage-encoded integrase with recombination occurring between specific attachment sites on the phage and mycobacterial chromosomes (attP and attB, respectively). Although some site-specific recombination systems simply involve binding of the recombinase to the sites of strand exchange, synapsis, and recombination, phage systems typically require the assembly of higher-order structures within which the recombinational potential of integrase is activated. The requirement for these structures derives from the necessity to regulate the directionality of recombination—either integration or excision—which must be closely coordinated with other aspects of the phage growth cycles. We show herein that there are multiple pathways available for the assembly of L5 recombination complexes, including the early synapsis of the attP and attB DNAs. This process is in contrast to the model for lambda integration and illustrates the different usage of molecular machineries to accomplish the same biological outcome.
Demonstrating in vivo interaction of two important
biomolecules and the relevance of the interaction to a biological
process have been difficult issues in biomedical research. Here, we
report the use of a homology modeling approach to establish the
significance of protein interactions in governing the activation of
programmed cell death in Caenorhabditis
elegans. A protein interaction cascade has been postulated to
mediate activation of cell death in nematodes, in which the
BH3-domain-containing (Bcl-2 homology region 3) protein EGL-1 binds the
cell-death inhibitor CED-9 and induces release of the death-activating
protein CED-4 from inhibitory CED-4/CED-9 complexes. We show here
that an unusual gain-of-function mutation in ced-9
(substitution of glycine 169 to glutamate) that results in potent
inhibition of most nematode cell deaths impairs the binding of EGL-1 to
CED-9 and EGL-1-induced release of CED-4 from CED-4/CED-9 complexes.
Based on a modeled EGL-1/CED-9 complex structure, we generated
second-site compensatory mutations in EGL-1 that partially restore the
binding of EGL-1 to CED-9(G169E) and EGL-1-induced release of CED-4
from CED-4/CED-9(G169E) complexes. Importantly, these mutations also
significantly suppress the death-protective activity of CED-9(G169E)
in vivo. These results establish that direct physical
interaction between EGL-1 and CED-9 is essential for the release of
CED-4 and the activation of cell death. The structure-based design of
second-site suppressors via homology modeling should be widely
applicable for probing important molecular interactions that are
implicated in fundamental biological processes.
We present a coupled two-way clustering approach to gene microarray
data analysis. The main idea is to identify subsets of the genes and
samples, such that when one of these is used to cluster the other,
stable and significant partitions emerge. The search for such subsets
is a computationally complex task. We present an algorithm, based on
iterative clustering, that performs such a search. This analysis is
especially suitable for gene microarray data, where the contributions
of a variety of biological mechanisms to the gene expression levels are
entangled in a large body of experimental data. The method was applied
to two gene microarray data sets, on colon cancer and leukemia. By
identifying relevant subsets of the data and focusing on them we were
able to discover partitions and correlations that were masked and
hidden when the full dataset was used in the analysis. Some of these
partitions have clear biological interpretation; others can serve to
identify possible directions for future research.
Circadian biological clocks control many biological events, but the pathways by which these events are controlled are largely unknown. Based on a model suggesting that cytosolic-free calcium levels control the expression of the Lhcb gene in plants, we tested whether the circadian oscillation of free calcium is responsible for driving the rhythm of Lhcb expression. We found that these rhythms free-run with different periods in tobacco seedlings in constant conditions. Moreover, robust oscillations of Lhcb promoter activity continued in undifferentiated tobacco calli in the absence of Ca2+ oscillations. Therefore, these two circadian rhythms are not linked hierarchically. These data provide evidence for separate circadian pacemakers controlling molecular events in plants.
In this study α-lactalbumin was converted from the regular, native state to a folding variant with altered biological function. The folding variant was shown to induce apoptosis in tumor cells and immature cells, but healthy cells were resistant to this effect. Conversion to HAMLET (human α-lactalbumin made lethal to tumor cells) required partial unfolding of the protein and a specific fatty acid, C18:1, as a necessary cofactor. Conversion was achieved with α-lactalbumin derived from human milk whey and with recombinant protein expressed in Escherichia coli. We thus have identified the folding change and the fatty acid as two key elements that define HAMLET, the apoptosis-inducing functional state of α-lactalbumin. Although the environment in the mammary gland favors the native conformation of α-lactalbumin that serves as a specifier in the lactose synthase complex, the conditions under which HAMLET was formed resemble those in the stomach of the nursing child. Low pH is known to release Ca2+ from the high-affinity Ca2+-binding site and to activate lipases that hydrolyze free fatty acids from milk triglycerides. We propose that this single amino acid polypeptide chain may perform vastly different biological functions depending on its folding state and the in vivo environment. It may be speculated that molecules like HAMLET can aid in lowering the incidence of cancer in breast-fed children by purging of tumor cells from the gut of the neonate.
Hominid brain size increased dramatically in the face of apparently severe associated evolutionary costs. This suggests that increasing brain size must have provided some sort of counterbalancing adaptive benefit. Several recent studies using magnetic resonance imaging (MRI) have indicated that a substantial correlation (mean r = ≈0.4) exists between brain size and general cognitive performance, consistent with the hypothesis that the payoff for increasing brain size was greater general cognitive ability. However, these studies confound between-family environmental influences with direct genetic/biological influences. To address this problem, within-family (WF) sibling differences for several neuroanatomical measures were correlated to WF scores on a diverse battery of cognitive tests in a sample of 36 sibling pairs. WF correlations between neuroanatomy and general cognitive ability were essentially zero, although moderate correlations were found between prefrontal volumes and the Stroop test (known to involve prefrontal cortex). These findings suggest that nongenetic influences play a role in brain volume/cognitive ability associations. Actual direct genetic/biological associations may be quite small, and yet still may be strong enough to account for hominid brain evolution.
Qureshi, Sajjad A.; Kim, Ronald M.; Konteatis, Zenon; Biazzo, Dawn E.; Motamedi, Haideh; Rodrigues, Robert; Boice, Judith A.; Calaycay, Jimmy R.; Bednarek, Maria A.; Griffin, Patrick; Gao, Ying-Duo; Chapman, Kevin; Mark, David F.
Fonte: The National Academy of SciencesPublicador: The National Academy of Sciences
Erythropoietin (EPO) controls the proliferation and differentiation of erythroid progenitor cells into red blood cells. EPO induces these effects by dimerization of the EPO receptors (EPOR) present on these cells. To discover nonpeptide molecules capable of mimicking the effects of EPO, we identified a small molecule capable of binding to one chain of EPOR and used it to synthesize molecules capable of inducing dimerization of the EPOR. We first identified compound 1 (N-3-[2-(4-biphenyl)-6-chloro-5-methyl]indolyl-acetyl-l-lysine methyl ester) by screening the in-house chemical collection for inhibitors of EPO binding to human EPOR and then prepared compound 5, which contains eight copies of compound 1 held together by a central core. Although both compounds inhibited EPO binding of EPOR, only compound 5 induced dimerization of soluble EPOR. Binding of EPO to its receptor in cells results in activation of many intracellular signaling molecules, including transcription factors like signal transducer and activator of transcription (STAT) proteins, leading to growth and differentiation of these cells. Consistent with its ability to induce dimerization of EPOR in solution, compound 5 exhibited much of the same biological activities as EPO...
Homologs of the chromatin-bound yeast silent information regulator 2 (SIR2) protein are found in organisms from all biological kingdoms. SIR2 itself was originally discovered to influence mating-type control in haploid cells by locus-specific transcriptional silencing. Since then, SIR2 and its homologs have been suggested to play additional roles in suppression of recombination, chromosomal stability, metabolic regulation, meiosis, and aging. Considering the far-ranging nature of these functions, a major experimental goal has been to understand the molecular mechanism(s) by which this family of proteins acts. We report here that members of the SIR2 family catalyze an NAD–nicotinamide exchange reaction that requires the presence of acetylated lysines such as those found in the N termini of histones. Significantly, these enzymes also catalyze histone deacetylation in a reaction that absolutely requires NAD, thereby distinguishing them from previously characterized deacetylases. The enzymes are active on histone substrates that have been acetylated by both chromatin assembly-linked and transcription-related acetyltransferases. Contrary to a recent report, we find no evidence that these proteins ADP-ribosylate histones. Discovery of an intrinsic deacetylation activity for the conserved SIR2 family provides a mechanism for modifying histones and other proteins to regulate transcription and diverse biological processes.
A major challenge in the study of gene regulation by NF-κB/Rel
transcription factors is to understand, at the biological and
mechanistic levels, the selective functions of individual Rel family
members. To study selectivity, we have examined the NF-κB/Rel
protein binding site (Rel site) within the IL-12 p40 promoter. IL-12 is
a proinflammatory cytokine expressed by activated macrophages that
serves as an essential inducer of T helper 1 cell development. In
nuclear extracts from lipopolysaccharideactivated macrophages, the
predominant Rel dimers capable of binding the IL-12 p40 Rel site were
the p50/p65 and p50/c-Rel heterodimers and p50/p50 homodimer. The
two heterodimers bound the site with comparable affinities and
exhibited comparable transactivation activities. In striking contrast,
p40 mRNA and protein concentrations were reduced dramatically in
c-Rel−/− macrophages and only modestly in
p65−/− macrophages. Other proinflammatory cytokine mRNAs
and proteins were not significantly reduced in c-Rel−/−
macrophages. These results reveal that a c-Rel-containing complex is an
essential and selective activator of p40 transcription, which may
reflect unique regulatory mechanisms or biological functions of IL-12.
The purpose of this paper is to propose certain dynamical principles in biological systems, which can be used to explain the effectiveness of charge transfer or excitation transfer in biological systems. Some of these systems are accessible experimentally.
Accurate localization of proteins within the substructure of cells and cellular organelles enables better understanding of structure–function relationships, including elucidation of protein–protein interactions. We describe the use of a near-field scanning optical microscope (NSOM) to simultaneously map and detect colocalized proteins within a cell, with superresolution. The system we elected to study was that of human red blood cells invaded by the human malaria parasite Plasmodium falciparum. During intraerythrocytic growth, the parasite expresses proteins that are transported to the erythrocyte cell membrane. Association of parasite proteins with host skeletal proteins leads to modification of the erythrocyte membrane. We report on colocalization studies of parasite proteins with an erythrocyte skeletal protein. Host and parasite proteins were selectively labeled in indirect immunofluorescence antibody assays. Simultaneous dual-color excitation and detection with NSOM provided fluorescence maps together with topography of the cell membrane with subwavelength (100 nm) resolution. Colocalization studies with laser scanning confocal microscopy provided lower resolution (310 nm) fluorescence maps of cross sections through the cell. Because the two excitation colors shared the exact same near-field aperture...
Peroxynitrite anion (ONOO−) is a reactive species of increasingly recognized biological relevance that contributes to oxidative tissue damage. At present, however, there is limited knowledge about the mechanisms of peroxynitrite diffusion through biological compartments. In this work we have studied the diffusion of peroxynitrite across erythrocyte membranes. In solution, peroxynitrite rapidly reacts with oxyhemoglobin to yield methemoglobin, with k2 = (10.4 ± 0.3) × 103 M−1⋅s−1 at pH 7.4 and 25°C. Addition of peroxynitrite to intact erythrocytes caused oxidation of intracellular oxyhemoglobin to methemoglobin. Oxidation yields in red blood cells at pH 7.0 were approximately 40% of those obtained in solution, which results mostly from competition of other cytosolic components for peroxynitrite. Indeed, rather small differences were observed between oxidation yields in lysates compared with intact erythrocytes, in particular at acidic and neutral pH values, indicating that membrane was not precluding peroxynitrite diffusion. Incubation of erythrocytes at pH 7.0 with 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), a specific inhibitor of anion exchange, resulted in up to 50% inhibition of oxyhemoglobin oxidation by peroxynitrite. More protection by DIDS was achieved at alkaline pH...
Cell surface oligosaccharides have been shown to play essential biological roles in such diverse biological phenomena as cellular adhesion, molecular recognition, and inflammatory response. The development of high-affinity ligands capable of selectively recognizing a variety of small motifs in different oligosaccharides would be of significant interest as experimental and diagnostic tools. As a step toward this goal we have developed DNA ligands that recognize the disaccharide cellobiose, whether in soluble form or as the repeating unit of the polymer, cellulose. These DNA “aptamers” bind with high selectivity to cellobiose with little or no affinity for the related disaccharides lactose, maltose, and gentiobiose. Thus, the DNA ligands can discriminate sugar epimers, anomers, and disaccharide linkages.
Methylglyoxal is an α-ketoaldehyde and dicarbonyl formed in cells as a side product of normal metabolism. Endogenously produced dicarbonyls, such as methylglyoxal, are involved in numerous pathogenic processes in vivo, including carcinogenesis and advanced glycation end-product formation; advanced glycation end-products are contributors to the pathophysiology of aging and chronic diabetes. Despite recent advances in understanding of the systemic effects of methylglyoxal, the full significance of this compound remains unknown. Herein we provide evidence that the majority of the methylglyoxal present in vivo is bound to biological ligands. The basis for our finding is an experimental approach that provides a measure of the bound methylglyoxal present in living systems, in this instance Chinese hamster ovary cells; with our approach, as much as 310 μM methylglyoxal was detected, 100- to 1,000-fold more than observed previously in biological systems. Several artifacts were considered before concluding that the methylglyoxal was associated with cellular structures, including phosphate elimination from triose phosphates, carbohydrate degradation under the assay conditions, and interference from the derivatizing agent used as part of the assay procedure. A major source of the recovered methylglyoxal is most probably modified cellular proteins. With methylglyoxal at about 300 μM...
The c-myb protooncogene is predominantly expressed in hematopoietic cells and plays a vital role in hematopoiesis. Retinoic acid (RA) is able to induce differentiation of several hematopoietic cells. This differentiation is linked to decreased c-myb expression, suggesting that retinoid receptors (RAR/RXR) may down-regulate c-myb gene expression. Furthermore, recent data indicate that RAR inhibits the function of the Myb protein itself. In addition, the Myb-Ets oncogenic fusion protein has been shown to inhibit transcriptional activation by RAR and thyroid hormone receptor. Myb-Ets also antagonizes the biological response of erythrocytic progenitor cells to RA and thyroid hormone. This prompted us to investigate a possible cross talk between RAR and Myb. Here, we demonstrate that RA inhibits the expression of the endogenous Myb target gene tom-1. Conversely, Myb functions as a potent inhibitor of RA-induced biological responses. Functional analysis of Myb mutants in transfection studies revealed that the Myb DNA-binding domain (DBD) is necessary for repression whereas the transactivation domain is dispensable. Furthermore, we show that v-Myb and RAR interact in vitro and in vivo. This interaction requires the DBD of RAR. In contrast...
Signal transducer and activator of transcription (STAT) proteins have been shown to mediate biological actions in response to cytokines. Stat3, a member of the STAT family, is activated by a variety of cytokines, including the interleukin 6 family of cytokines, leptin, granulocyte colony-stimulating factor, and epidermal growth factor. To address the biological function of Stat3, we generated mice deficient in Stat3 by gene targeting. No viable Stat3-deficient mice could be obtained from heterozygote intercross. Analysis of embryos at several gestation times revealed that Stat3-deficient embryos showed a rapid degeneration between embryonic days 6.5 and 7.5, although they developed into the egg cylinder stage until embryonic day 6.0. These results demonstrate that Stat3 is essential for the early development of mouse embryos.
A novel method was developed to detect molecular associations of dystrophin with actin in cryostat muscle tissue sections by combining resonance energy transfer technology with immunohistochemical techniques. This method takes advantage of the long phosphorescent lifetime of terbium chelates, a property that enables the accurate determination of energy transfer in biological tissues by lifetime measurements of sensitized emission. After a brief excitation pulse, terbium chelates emit for milliseconds after the intrinsically high autofluorescence of biological specimens has decayed to negligible levels. Rat skeletal muscle tissue sections were labeled with both anti-dystrophin monoclonal antibody conjugated to a terbium-based resonance energy transfer donor and anti-actin tetramethylrhodamine phalloidin as an acceptor. Resonance energy transfer between the two probes indicated that the distance separating the probes is within 10 nm (about the size of an IgG2b antibody molecule). The fraction of antibodies that participated in resonance energy transfer was estimated to be 80–90% because of the close agreement between the quenching of donor phosphorescence and the efficiency of resonance energy transfer revealed by lifetime measurements of sensitized emission by tetramethyl-rhodamine phalloidin. Sensitized emission was detectable only when both anti-dystrophin antibody and tetramethyl-rhodamine phalloidin were present. These results indicate that actin and dystrophin are closely associated within the cell. This method is potentially applicable to the investigation of many types of intracellular associations.
Receptor tyrosine kinases Flt-1 and Flk-1/KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1/KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice (flt-1TK−/−) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in flt-1TK−/− mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.