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PubMed2006

Characterization of the backbone and side chain dynamics of the CaM-CaMKIp complex reveals microscopic contributions to protein conformational entropy.

Calmodulin is a central mediator of calcium-dependent signal transduction pathways and regulates the activity of a large number of diverse targets. Calcium-dependent interactions of calmodulin with regulated proteins are of generally high affinity but of quite variable thermodynamic origins. Here we investigate the influence of the binding of the calmodulin-binding domain of calmodulin kinase I on the fast internal dynamics of calcium-saturated calmodulin. NMR relaxation was used to probe motion on the backbone (viewed through the backbone amide NH group) and the side chains (viewed through methyl groups). The distribution of the amplitudes of side chain dynamics is trimodal. The microscopic details of side chain motion are compared with those of a thermodynamically and structurally similar complex of calmodulin with the calmodulin-binding domain of the smooth muscle myosin light chain kinase. While there are no significant differences in backbone dynamics and no net change in methyl-bearing side chain dynamics, a large redistribution of the amplitude of methyl dynamics is observed between the two complexes. The variation in dynamics was largely localized to the heterogeneously dynamic target-binding interface, suggesting that differential dynamics of the binding surface plays a functional role in the high-affinity binding interactions of calmodulin. These results begin to reveal a fundamental role for residual protein entropy in molecular recognition by calmodulin.

Amino Acid Sequence↗
PubMed1997

A mixed-charge pair in human interleukin 4 dominates high-affinity interaction with the receptor alpha chain.

Human interleukin 4 (IL-4) binds to its cellular receptor with a Kd in the subnanomolar range, similar to many other 4-helix-bundle proteins interacting with members of the hematopoietin (cytokine) receptor superfamily. In the IL-4 system this interaction is predominantly determined by the extracellular domain (IL4-BP) of the receptor alpha chain (Kd approximately 150 pM). Now a high-resolution mutational and kinetic analysis has revealed that the high-affinity binding of IL-4 originates from a continuous patch of a few mostly polar or charged amino acid side chains located on helices A and C. The binding epitope comprises (i) a set of side chains determining the dissociation rate (k(off)) and (ii) a partially overlapping set determining the association rate constant (k(on)) of the IL-4/IL4-BP complex. The k(off) epitope is assembled from two juxtaposed main determinants (Glu-9 and Arg-88) surrounded by five side chains (Ile-5, Thr-13, Arg-53, Asn-89, and Trp-91) of lower importance. The cumulative increase in k(off) after alanine substitution is 10(5)-fold for the central mixed-charge pair and 3 x 10(3)-fold for the satellites. The k(on) epitope is formed by five positively charged residues on helix C (Lys-77, Arg-81, Lys-84, Arg-85, and Arg-88) and two neighboring residues on helix A (Glu-9 and Thr-13). The cumulative loss in k(on) of the alanine variants is only about 10-fold. These results provide the basis for an understanding of molecular recognition in cytokine receptor complexes and for an IL-4 antagonist design.

Alanine↗
PubMed1988

Nuclear lamina heterogeneity in mammalian cells. Differential expression of the major lamins and variations in lamin B phosphorylation.

We have studied the molecular composition of the nuclear lamina in rat tissues of distinct embryological origin and the occurrence of the nuclear lamins during in vitro differentiation of the mouse F9 teratocarcinoma cell line. Immunochemical analysis demonstrated that all rat tissues contained the three major lamin forms (lamins A, B, and C) previously recognized in rat liver nuclei; however, other minor cross-reactive components were also identified in some tissues. The amount of the 67-kDa lamin B complexed with lamins A and C in the laminae of different tissues ranged from a stoichiometry of much less than 1 to approximately 1. Furthermore, it was found that F9 stem cells and their differentiated progeny express only lamin B, and Northern blotting analysis indicated that these cells fail to accumulate lamin A and C mRNA. Chemical cleavages and peptide mapping suggested that the 67-kDa lamin B form was of similar primary structure in all differentiated tissues and F9 cells. Employing antibodies with different affinities for phosphorylated and nonphosphorylated lamin B, we showed that the apparent invariance in the expression of this polypeptide is overriden by a heterogeneity produced via tissue-specific phosphorylation. Because similar differences in antibody recognition could be reproduced in vitro by phosphorylating lamin B with protein kinase A, we have concluded that the tissue-specific modifications of this protein may occur at consensus sites recognized by this enzyme. These data support the hypotheses that the lamins can form functional laminae by associating at various combinations, and that processes including differential lamin synthesis and post-translational modification can produce a steady state lamina heterogeneity.

Animals↗
PubMed1994

Role of two operators in regulating the plasmid-borne raf operon of Escherichia coli.

The plasmid-borne raf operon encodes functions required for the inducible uptake and utilization of raffinose in Escherichia coli K12. The expression of three structural genes for alpha-galactosidase (rafA), Raf permease (rafB) and sucrose hydrolase (rafD) is negatively controlled by the binding of RafR repressor (rafR) to two operator sites, O1 and O2, that flank the -35 sequence of the raf promoter, PA. In vitro, O1 and O2 are occupied on increasing the concentration of RafR, without detectable preference for one site or the other or any indication of cooperative binding. Nucleotide substitutions at positions 3, 4 or 5 in an operator half-site prevented repressor binding, supporting a model that postulates specific interactions of these base pairs with the recognition helix of RafR. To study the role of each operator site, we have compared by gel shift analysis the binding of purified RafR repressor to DNA fragments containing the original O1O2 configuration or mutant O1 or O2. When either one of the two operators was inactivated by site-directed mutagenesis, both O1 and O2 exhibited the same affinity for repressor and the same sensitivity to arrest of repressor binding by the natural inducer, melibiose. However, in the native O1O2 configuration, simultaneous binding of RafR to both operators was sterically hindered, leading to a 13-fold decrease in the intrinsic affinity of an operator site for repressor, once the other site had been occupied. To assess the role of each operator in vivo, rafA was used as a reporter gene. A 1200-fold repression (100%) was exerted by RafR binding to the native O1O2 configuration, whereas O2 alone exerted 45% and O1 alone 6% repression of rafA transcription. The differential effects of O1 versus O2 on transcription (despite matching affinities of O1 and O2 for repressor) suggest that positioning of the O2-repressor complex between the -35 and -10 signals is crucial for transcription control and that repressor binding to the upstream O1 serves to enhance this effect.

Autoradiography↗
PubMed2003

c-MIR, a human E3 ubiquitin ligase, is a functional homolog of herpesvirus proteins MIR1 and MIR2 and has similar activity.

Kaposi's sarcoma associated-herpes virus encodes two proteins, MIR (modulator of immune recognition) 1 and 2, which are involved in the evasion of host immunity. MIR1 and 2 have been shown to function as an E3 ubiquitin ligase for immune recognition-related molecules (e.g. major histocompatibility complex class I, B7-2, and ICAM-1) through the BKS (bovine herpesvirus 4, Kaposi's sarcoma associated-herpes virus, and Swinepox virus) subclass of plant homeodomain (PHD) domain, termed the BKS-PHD domain. Here we show that the human genome also encodes a novel BKS-PHD domain-containing protein that functions as an E3 ubiquitin ligase and whose putative substrate is the B7-2 co-stimulatory molecule. This novel E3 ubiquitin ligase was designated as c-MIR (cellular MIR) based on its functional and structural similarity to MIR1 and 2. Forced expression of c-MIR induced specific down-regulation of B7-2 surface expression through ubiquitination, rapid endocytosis, and lysosomal degradation of the target molecule. This specific targeting was dependent upon the binding of c-MIR to B7-2. Replacing the BKS-PHD domain of MIR1 with the corresponding domain of c-MIR did not alter MIR1 function. The discovery of c-MIR, a novel E3 ubiquitin ligase, highlights the possibility that viral immune regulatory proteins originated in the host genome and presents unique functions of BKS-PHD domain-containing proteins in mammals.

Amino Acid Sequence↗
PubMed2004

Species boundaries, phylogeography and conservation genetics of the red-legged frog (Rana aurora/draytonii) complex.

The red-legged frog, Rana aurora, has been recognized as both a single, polytypic species and as two distinct species since its original description 150 years ago. It is currently recognized as one species with two geographically contiguous subspecies, aurora and draytonii; the latter is protected under the US Endangered Species Act. We present the results of a survey of 50 populations of red-legged frogs from across their range plus four outgroup species for variation in a phylogenetically informative, approximately 400 base pairs (bp) fragment of the mitochondrial cytochrome b gene. Our mtDNA analysis points to several major results. (1) In accord with several other lines of independent evidence, aurora and draytonii are each diagnosably distinct, evolutionary lineages; the mtDNA data indicate that they do not constitute a monophyletic group, but rather that aurora and R. cascadae from the Pacific northwest are sister taxa; (2) the range of the draytonii mtDNA clade extends about 100 km further north in coastal California than was previously suspected, and corresponds closely with the range limits or phylogeographical breaks of several codistributed taxa; (3) a narrow zone of overlap exists in southern Mendocino County between aurora and draytonii haplotypes, rather than a broad intergradation zone; and (4) the critically endangered population of draytonii in Riverside County, CA forms a distinct clade with frogs from Baja California, Mexico. The currently available evidence favours recognition of aurora and draytonii as separate species with a narrow zone of overlap in northern California.

Animals↗
PubMed1995

Carrier-reactive hapten-specific cytotoxic T lymphocyte clones originate from a highly preselected T cell repertoire: implications for chemical-induced self-reactivity.

We have recently described trinitrophenyl (TNP)-specific cytotoxic T lymphocyte (CTL) clones from C57BL/6 mice specific for hapten-modified peptides bearing a TNP-lysine in a peripheral position, i.e. in position 7 of H-2Kb-bound octapeptides. CTL recognition of such determinants is always sequence-dependent due to co-recognition of TNP as well as amino acid side chains of the carrier peptide. By the use of glycine-based designer peptides for primary induction of CTL in vitro, we have identified two sub-epitopes on individual position 7-haptenated peptides that form two TcR contact points and which can be independently recognized by cloned CTL. One of these sub-epitopes is represented by the hapten itself, the other by the amino acids tyrosine and lysine in positions 3 and 4 of the carrier peptide, respectively. Immunization with such TNP-modified peptides frequently results in the specific induction of CTL also reacting with the unmodified carrier peptides. DNA sequence analyses of the TcR revealed an extraordinary similarity of several independent TcR of CTL from individual mice and induced with different TNP-peptides. These receptor similarities clearly correlate with structural elements common to the immunizing peptides and suggest their origin from positive thymic selection of TcR on Kb-associated associated self-peptides bearing Tyr in position 3. Our data provide additional information concerning the topology of TcR binding to peptide/MHC complexes with, but also without, TNP. They also indicate a mechanism which might explain the potential of chemicals or drugs to induce autoimmune phenomena.

Amino Acid Sequence↗
PubMed2001

Study of gas-phase molecular recognition using Fourier transform ion cyclotron resonance mass spectrometry (FTICR/MS).

The application of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to the quantitative study of molecular recognition in the gas phase is reviewed. Because most quantitative measurements are dependent on accurate determination of the pressure of a neutral reagent, methods for accurate pressure measurement in FTICR, including gauge calibration using a reaction with known rate constants (the traditional method), exothermic proton transfer rate measurement (often the best method when accurate neutral pressures in the trapping cell are desired), and linewidth measurement (a little-used, but generally applicable method) are discussed. The use of rate constant measurements in molecular recognition is illustrated with examples employing natural abundance isotopic labeling to study self-exchange and 2 : 1 ligand:metal complex formation kinetics in crown ether-alkali cation systems. Self-exchange rates do not correlate with alkali cation/crown cavity size relationships, whereas 2 : 1 complex formation kinetics correlate strongly with size relationships. The use of exchange equilibrium constant measurements to characterize molecular recognition is illustrated by alkali cation exchanges between 18-crown-6 and the isomers of dicyclohexano-18-crown-6. These experiments show that the alkyl-substituted ligand binds alkali cations better than unsubstituted 18-crown-6 in the gas phase, in accordance with expectations based on the higher polarizability of the alkyl-substituted ligand. Further, the metal binding thermochemistry differs for the two dicyclohexano-18-crown-6 isomers, with the bowl-shaped cis-syn-cis isomer binding all the alkali cations more strongly than the cis-anti-cis isomer. The measurement of entropies and enthalpies associated with one of the most subtle forms of molecular recognition, enantiomeric discrimination, is illustrated by studies of the discrimination between enantiomers of chiral amines by dimethyldiketopyridino-18-crown-6. This chiral ligand binds chiral primary ammonium cations that have the opposite absolute configuration at their stereocenter more strongly than the enantiomer with the same absolute configuration. Gas-phase studies show that this enantiomeric discrimination is enthalpic in origin, likely related to more favorable pi-pi stacking for the preferred enantiomer. Entropy disfavors binding of the preferred enantiomer.

Journal Article↗
PubMed2005

Structural evidence of a passive base-flipping mechanism for AGT, an unusual GT-B glycosyltransferase.

The Escherichia coli T4 bacteriophage uses two glycosyltransferases to glucosylate and thus protect its DNA: the retaining alpha-glucosyltransferase (AGT) and the inverting beta-glucosyltransferase (BGT). They glucosylate 5-hydroxymethyl cytosine (5-HMC) bases of duplex DNA using UDP-glucose as the sugar donor to form an alpha-glucosidic linkage and a beta-glucosidic linkage, respectively. Five structures of AGT have been determined: a binary complex with the UDP product and four ternary complexes with UDP or UDP-glucose and oligonucleotides containing an A:G, HMU:G (hydroxymethyl uracyl) or AP:G (apurinic/apyrimidinic) mismatch at the target base-pair. AGT adopts the GT-B fold, one of the two folds known for GTs. However, while the sugar donor binding mode is classical for a GT-B enzyme, the sugar acceptor binding mode is unexpected and breaks the established consensus: AGT is the first GT-B enzyme that predominantly binds both the sugar donor and acceptor to the C-terminal domain. Its active site pocket is highly similar to four retaining GT-B glycosyltransferases (trehalose-6-phosphate synthase, glycogen synthase, glycogen and maltodextrin phosphorylases) strongly suggesting a common evolutionary origin and catalytic mechanism for these enzymes. Structure-guided mutagenesis and kinetic analysis do not permit identification of a nucleophile residue responsible for a glycosyl-enzyme intermediate for the classical double displacement mechanism. Interestingly, the DNA structures reveal partially flipped-out bases. They provide evidence for a passive role of AGT in the base-flipping mechanism and for its specific recognition of the acceptor base.

Bacteriophage T4↗
PubMed1988

Intermolecular interactions between protein and other molecules including hydration effects.

The structural aspects of protein functions, e.g., molecular recognition such as enzyme-substrate and antibody-antigen interactions, are elucidated in terms of dehydration and atomic interactions. When a protein interacts with some target molecule, water molecules at the interacting regions of both molecules are removed, with loss of the hydration free energy, but gaining atomic interactions between atoms of the contact sites in both molecules. The free energies of association originating from the dehydration and interactions between the atoms can be computed from changes in the accessible surface areas of the atoms involved. The free energy due to interactions between atomic groups at the contact sites is estimated as the sum of those estimated from the changes in the accessible surface area of 7 atomic groups, assuming that the interactions are proportional to the change of the area. The chain enthalpies and entropies evaluated from experimental thermodynamic properties and hydration quantities at the standard temperature for 10 proteins were available to determine the proportional constants for the atomic groups. This method was applied to the evaluation of association constants for the dimerization of proteins and the formation of proteolytic enzyme-inhibitor complexes, and the computed constants were in agreement with the experimental ones. However, the method is not accurate enough to account quantitatively for the change in the thermal stability of mutants of T4 lysozyme. Nevertheless, this method provides a way to elucidate the interactions between molecules in solution.

Proteins↗
PubMed2006

Evolving concepts in pheochromocytoma and paraganglioma.

PURPOSE OF REVIEW: The pheochromocytoma field has recently undergone a paradigm shift. This review will highlight some of these novel findings, including their impact on our understanding of the disease biology and influence on clinical management. RECENT FINDINGS: Identification of novel susceptibility loci and recognition of a high rate of germline mutations in pheochromocytomas indicate that their genetic diversity is broader and more complex than previously estimated. Further, increased risk of tumor malignancy and aggressiveness in certain patients with succinate dehydrogenase subunit B(SDHB) mutations suggest that they may have prognostic value as predictors of pheochromocytoma behavior. Finally, discovery of a shared activation of the hypoxic response in pheochromocytomas with mutations in VHL and SDH genes and uncovering of a common JunB-mediated apoptosis defect in the major hereditary groups of pheochromocytoma have provided a mechanistic basis for the clinical similarities between these distinct syndromes. SUMMARY: The notion that 'sporadic'-appearing tumors may in fact be components of one of multiple hereditary syndromes has a major impact on surveillance and follow-up of patients and their at-risk family members. Likewise, the ability to predict tumor malignancy has the potential to improve the prognosis of these patients. Importantly, insights into the biology of pheochromocytomas have provided clues on pathway interactions in cancers and have laid the ground for generation of new hypotheses on the cell-of-origin of these tumors. Pheochromocytomas have therefore emerged as key models for understanding cancer biology and for paving the way for future designer treatment in this and other cancers.

Biomarkers, Tumor↗
PubMedDate not supplied

A Beauveria phylogeny inferred from nuclear ITS and EF1-alpha sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs.

Beauveria is a globally distributed genus of soil-borne entomopathogenic hyphomycetes of interest as a model system for the study of entomopathogenesis and the biological control of pest insects. Species recognition in Beauveria is difficult due to a lack of taxonomically informative morphology. This has impeded assessment of species diversity in this genus and investigation of their natural history. A gene-genealogical approach was used to investigate molecular phylogenetic diversity of Beauveria and several presumptively related Cordyceps species. Analyses were based on nuclear ribosomal internal transcribed spacer (ITS) and elongation factor 1-alpha (EF1-alpha) sequences for 86 exemplar isolates from diverse geographic origins, habitats and insect hosts. Phylogenetic trees were inferred using maximum parsimony and Bayesian likelihood methods. Six well supported clades within Beauveria, provisionally designated A-F, were resolved in the EF1-alpha and combined gene phylogenies. Beauveria bassiana, a ubiquitous species that is characterized morphologically by globose to subglobose conidia, was determined to be non-monophyletic and consists of two unrelated lineages, clades A and C. Clade A is globally distributed and includes the Asian teleomorph Cordyceps staphylinidaecola and its probable synonym C. bassiana. All isolates contained in Clade C are anamorphic and originate from Europe and North America. Clade B includes isolates of B. brongniartii, a Eurasian species complex characterized by ellipsoidal conidia. Clade D includes B. caledonica and B. vermiconia, which produce cylindrical and comma-shaped conidia, respectively. Clade E, from Asia, includes Beauveria anamorphs and a Cordyceps teleomorph that both produce ellipsoidal conidia. Clade F, the basal branch in the Beauveria phylogeny includes the South American species B. amorpha, which produces cylindrical conidia. Lineage diversity detected within clades A, B and C suggests that prevailing morphological species concepts underestimate species diversity within these groups. Continental endemism of lineages in B. bassiana s.l. (clades A and C) indicates that isolation by distance has been an important factor in the evolutionary diversification of these clades. Permutation tests indicate that host association is essentially random in both B. bassiana s.l. clades A and C, supporting past assumptions that this species is not host specific. In contrast, isolates in clades B and D occurred primarily on coleopteran hosts, although sampling in these clades was insufficient to assess host affliation at lower taxonomic ranks. The phylogenetic placement of Cordyceps staphylinidaecola/bassiana, and C. scarabaeicola within Beauveria corroborates prior reports of these anamorph-teleomorph connections. These results establish a phylogenetic framework for further taxonomic, phylogenetic and comparative biological investigations of Beauveria and their corresponding Cordyceps teleomorphs.

Animals↗
PubMed1992

Surface expression of functional IgE binding protein, an endogenous lectin, on mast cells and macrophages.

IgE-binding protein (epsilon BP) is a galactoside-specific lectin containing an S-type carbohydrate-recognition domain. It was originally identified in rat basophilic leukemia cells and is now known to be identical to a macrophage surface Ag, Mac-2, and lectins designated as CBP 35/L-34/RL-29. It has also been related to a nonintegrin laminin-binding protein isolated from mouse macrophages. In this report we have shown the following: epsilon BP is present in variable amounts in several mast cell lines, and the surface expression of epsilon BP in these cell lines is quite variable and does not correlate with the total amount of epsilon BP in the cell. epsilon BP is displayed on the cell surface in a manner that is reversible by lactose, most likely through attachment to yet unidentified glycoconjugates. The putative epsilon BP binding sites on the cell surface can be readily demonstrated by using radiolabeled epsilon BP, and the sites are present in comparable amounts in various cell lines. Expression of epsilon BP on the cell surface can be regulated; the most notable example is the upregulation of surface epsilon BP on RBL cells activated through the high-affinity IgE receptor by IgE immune complexes. Cell-surface epsilon BP is functional as measured by its ability to promote adhesion of trypsinized rabbit erythrocytes to mast cells and macrophages. On the basis of these results and reported properties of related lectins, we propose that the lectin represented by epsilon BP is a new class of cell-adhesion protein.

Animals↗
PubMed1998

Structural recognition and distortion by the DNA junction-resolving enzyme RusA.

RusA is a relatively small DNA junction-resolving enzyme of lambdoid phage-origin. Many of the physical characteristics of this enzyme are similar to those of junction-resolving enzymes of different origins. RusA binds to DNA junctions as a dimer, with a dissociation constant of 2 to 7 nM. RusA also exists in dimeric form in free solution, with a half time for subunit exchange of 4.2 minutes. We find that RusA can cleave both fixed junctions and those that can undergo a number of steps of branch migration, and confirm that the enzyme exhibits a strong preference for cleavage 5' to a CpC sequence. We have isolated a mutant protein, RusA D70N, that is completely inactive in cleavage while binding normally to DNA junctions, suggesting a role for aspartate 70 in the cleavage reaction. Constraining the conformation of the junction by means of tethering the helical ends leads to a marked reduction in cleavage rate by RusA, suggesting that the structure must be altered for cleavage. Using comparative gel electrophoresis we find that the global structure of the DNA junction is altered on RusA binding, into a structure that is different from any that is formed by the free junction. Moreover, the structure of the complex is the same irrespective of the presence or absence of magnesium ions. Thus, like all the junction-resolving enzymes, RusA both recognises and distorts the structure of DNA junctions.

Catalysis↗
PubMed2002

Initiation of DNA replication in eukaryotes is an intriguing cascade of protein interactions.

Initiation of eukaryotic DNA replication is a complex process including the recognition of initiation sites on DNA, multi-step DNA preparation for duplication, and assembly of multi-protein complexes capable of beginning DNA synthesis at initiation sites. The process starts at the late M phase and lasts till the appropriate time of the S phase for each initiation site. A chain of interesting interactions between Orc1p-6p, Cdc6p, Mcm2p-7p, Mcm10p, Cdt1, Cdc45p, Dbf4/Cdc7p, RPA, and DNA polymerase alpha takes place during this period. The sequence of these interactions is controlled by cyclin-dependent kinases, as well as by ubiquitin-dependent proteolysis in the proteasome. This review summarizes the data on proteins initiating DNA replication and factors controlling their activities.

Binding Sites↗
PubMed2002

Lack of IFN-gamma-mediated induction of the class II transactivator (CIITA) through promoter methylation is predominantly found in developmental tumor cell lines.

Downregulation of major histocompatibility complex (MHC) molecules by tumor cells impairs cellular immune recognition and contributes to inefficient cell-mediated tumor eradication. Low or lack of expression of MHC molecules is frequently observed in early developmental or embryonically derived tumor cells. Considering the central role of the class II transactivator (CIITA) in MHC class II- and class I-mediated antigen presentation, we compared the induction of CIITA by interferon-gamma (IFN-gamma) in a diverse panel of developmental and more differentiated tumor cell lines. In contrast to the more differentiated tumor cell lines, none of the developmental tumor cell lines were capable of expressing CIITA after treatment with IFN-gamma. Remarkably, in transient transfection assays, CIITA promoter IV (CIITA-PIV) was found to be induced by IFN-gamma. Southern blot analysis of genomic DNA obtained from the developmental tumor cell lines indicated that the absence of endogenous CIITA induction was due to methylation of the CIITA-PIV region. Exposure to 5-azacytidine restored induction of CIITA and congruent HLA-DRA expression in these cells. The observation that only developmental tumor cell lines, originating from various tissues, employ methylation to silence CIITA expression may reflect the natural status of CIITA expression during early development rather than oncogenic transformation.

Azacitidine↗
PubMed2006

Fast point-based 3-D alignment of live cells.

Typical time intervals between acquisitions of three-dimensional (3-D) images of the same cell in live cell imaging are in the orders of minutes. In the meantime, the live cell can move in a water basin on the stage. This movement can hamper the studies of intranuclear processes. We propose a fast point-based image registration method for the suppression of the movement of a cell as a whole in the image data. First, centroids of certain intracellular objects are computed for each image in a time-lapse series. Then, a matching between the centroids, which have the maximal number of pairs, is sought between consecutive point sets by a 3-D extension of a two-dimensional fast point pattern matching method, which is invariant to rotation, translation, local distortion, and extra/missing points. The proposed 3-D extension assumes rotations only around the z axis to retain the complexity of the original method. The final step involves computing the optimal fully 3-D transformation between images from corresponding points in the least-squares manner. The robustness of the method was evaluated on generated data. The results of the simulations show that the method is very precise and its correctness can be estimated. This article also presents two practical application examples, namely the registration of images of HP1 domains and the registration of images of telomeres. More than 97% of time-consecutive images were successfully registered. The results show that the method is very well suited to live cell imaging.

Algorithms↗
PubMed1982

Pharmacology of GABA.

GABA-ergic systems are involved in all the main functions of the brain. In most brain regions impairment of this system produces epileptic activity. GABA-mediated inhibitory function can be enhanced by drugs of at least seven different types. They act on the metabolism or synaptic release of GABA, or its reuptake into neurones of glia, or on various components of the GABA receptor complex (GABA recognition site, "benzodiazepine" receptor or chloride ionophore). Among such compounds, those which act most specifically and potently on GABA receptors remain primarily research tools. Among compounds in clinical use, valproate, benzodiazepines, and anticonvulsant barbiturates al enhance GABA-mediated inhibition. In the future, new inhibitors of GABA uptake, new GABA agonists and potent inhibitors of GABA-transaminase are likely to become available. Trials of drugs enhancing GABA-ergic function have been made in a wide variety of neurological disorders. In most forms of epilepsy a therapeutic effect is evident. Real benefit from GABA therapies has not been demonstrated in the principal disorders of movement (Huntington's chorea, Parkinson's disease, dystonias), except in so far as they have a myoclonic or paroxysmal component. Among psychiatric disorders the acute symptoms of schizophrenia are exacerbated by enhanced GABA-ergic function. Abstinence syndromes (alcohol, barbiturate or narcotic withdrawal) are ameliorated by drugs enhancing GABA-ergic function, and there is some evidence for a beneficial action in anxiety states and mania. Attempts to relate the molecular neurobiology of GABA with clinical pharmacology are of very recent origin. Improved understanding of the variety of GABA receptor mechanisms will provide the key to the more selective pharmacological manipulations that are required for therapeutic success.

4-Aminobutyrate Transaminase↗