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.
The Hrp (type III protein secretion) system is essential for the plant parasitic ability of Pseudomonas syringae and most Gram-negative bacterial plant pathogens. AvrB and AvrPto are two P. syringae proteins that have biological activity when produced via heterologous gene expression inside plant cells or when produced by Hrp+ bacteria. Avr-like proteins, presumably injected by the Hrp system on bacterial contact with plant cells, appear to underlie pathogenic interactions, but none has been observed outside of the bacterial cytoplasm, and identifying novel genes encoding them is tedious and uncertain without a phenotype in culture. Here we describe a cloned Hrp secretion system that functions heterologously in Escherichia coli to secrete AvrB and AvrPto in culture and to promote AvrB and AvrPto biological activity in inoculated plants. The hrp gene cluster, carried on cosmid pCPP2156, was cloned from Erwinia chrysanthemi, a pathogen that differs from P. syringae in being host promiscuous. E. coli DH5α carrying pCPP2156, but not related Hrp-deficient cosmids, elicited a hypersensitive response in Nicotiana clevelandii only when also expressing avrB in trans. The use of pAVRB-FLAG2 and pAVRPTO-FLAG, which produce Avr proteins with a C-terminal FLAG-epitope fusion...
SR protein kinases (SRPKs) and their substrates, the SR family of serine/arginine-rich pre-mRNA splicing factors, appear to be key regulators of alternative splicing. Although SR proteins have been well characterized through biochemical experiments in metazoans, their functions in vivo are unclear. Because of the strict splice site consensus and near absence of alternative splicing in Saccharomyces cerevisiae, it had been thought that budding yeast would lack an SRPK and its substrates. Here, we present structural, biochemical, and cell-biological evidence that directly demonstrates an SR protein kinase, Sky1p, as well as a number of SRPK substrates in S. cerevisiae. One of these substrates is Npl3p, an SR-like protein involved in mRNA export. This finding raises the provocative possibility that Sky1p, and by extension metazoan SRPKs, regulates mRNA export or the nucleocytoplasmic shuttling of RS domain proteins. The unexpected discovery of an SR protein kinase in budding yeast provides a foundation for genetic dissection of the biological functions of SR proteins and their kinases.
Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation in man. Genes containing one or more SNPs can give rise to two or more allelic forms of mRNAs. These mRNA variants may possess different biological functions as a result of differences in primary or higher order structures that interact with other cellular components. Here we report the observation of marked differences in mRNA secondary structure associated with SNPs in the coding regions of two human mRNAs: alanyl tRNA synthetase and replication protein A, 70-kDa subunit (RPA70). Enzymatic probing of SNP-containing allelic fragments of the mRNAs revealed pronounced allelic differences in cleavage pattern at sites 14 or 18 nt away from the SNP, suggesting that a single-nucleotide variation can give rise to different mRNA folds. By using phosphorothioate oligodeoxyribonucleotides complementary to the region of different allelic structures in the RPA70 mRNA, but not extending to the SNP itself, we find that the SNP exerts an allele-specific effect on the accessibility of its flanking site in the endogenous human RPA70 mRNA. This further supports the allele-specific structural features identified by enzymatic probing. These results demonstrate the contribution of common genetic variation to structural diversity of mRNA and suggest a broader role than previously thought for the effects of SNPs on mRNA structure and...
A genetic annealing model for the universal ancestor of all extant life is presented; the name of the model derives from its resemblance to physical annealing. The scenario pictured starts when “genetic temperatures” were very high, cellular entities (progenotes) were very simple, and information processing systems were inaccurate. Initially, both mutation rate and lateral gene transfer levels were elevated. The latter was pandemic and pervasive to the extent that it, not vertical inheritance, defined the evolutionary dynamic. As increasingly complex and precise biological structures and processes evolved, both the mutation rate and the scope and level of lateral gene transfer, i.e., evolutionary temperature, dropped, and the evolutionary dynamic gradually became that characteristic of modern cells. The various subsystems of the cell “crystallized,” i.e., became refractory to lateral gene transfer, at different stages of “cooling,” with the translation apparatus probably crystallizing first. Organismal lineages, and so organisms as we know them, did not exist at these early stages. The universal phylogenetic tree, therefore, is not an organismal tree at its base but gradually becomes one as its peripheral branchings emerge. The universal ancestor is not a discrete entity. It is...
The nontoxic proteolytic C fragment of tetanus toxin (TTC peptide) has the same ability to bind nerve cells and be retrogradely transported through a synapse as the native toxin. We have investigated its potential use as an in vivo neurotropic carrier. In this work we show that a hybrid protein encoded by the lacZ–TTC gene fusion retains the biological functions of both proteins in vivo—i.e., retrograde transynaptic transport of the TTC fragment and β-galactosidase enzymatic activity. After intramuscular injection, enzymatic activity could be detected in motoneurons and connected neurons of the brainstem areas. This strategy could be used to deliver a biological activity to neurons from the periphery to the central nervous system. Such a hybrid protein could also be used to map synaptic connections between neural cells.