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PubMed2001

Self-priming of retroviral minus-strand strong-stop DNAs.

After minus-strand strong-stop DNA (-sssDNA) is synthesized, the RNA template is degraded by the RNase H activity of the reverse transcriptase (RT), generating a single-stranded DNA. The 3' end of -sssDNA from HIV-1 can form a hairpin; this hairpin will self-prime in vitro. We previously used a model substrate, -R ssDNA, which corresponds to the 3' end of the -sssDNA of HIV-1, to show that the self-priming of this model substrate could be prevented by annealing a 17-nt-long DNA oligonucleotide to the 3' end of -R ssDNA in the presence of HIV-1 nucleocapsid (NC) protein. Similar model substrates were prepared for HIV-2 and HTLV-1; the R regions of these two viruses are longer and form more complex structures than the R region of the HIV-1 genome. However, the size of the R region and the complexity of the secondary structures they can form do not affect self-priming or its prevention. The efficiency of the self-priming is related to the relative stabilities of the conformations of -R ssDNA that can and cannot induce self-priming. For the three viruses (HIV-1, HIV-2, and HTLV-1), the size of the DNA oligonucleotide needed to block self-priming in the presence of NC is similar to the expected size of the piece of RNA left after degradation of the RNA template during reverse transcription. We also found that when the 3' end of -R ssDNA is annealed to a complementary DNA oligonucleotide, it is a good substrate for efficient nonspecific strand transfer to other single-stranded DNA molecules.

Base Sequence↗

PubMed2005

Schema-based learning of adaptable and flexible prey- catching in anurans II. Learning after lesioning.

The previous companion paper describes the initial (seed) schema architecture that gives rise to the observed prey-catching behavior. In this second paper in the series we describe the fundamental adaptive processes required during learning after lesioning. Following bilateral transections of the hypoglossal nerve, anurans lunge toward mealworms with no accompanying tongue or jaw movement. Nevertheless anurans with permanent hypoglossal transections eventually learn to catch their prey by first learning to open their mouth again and then lunging their body further and increasing their head angle. In this paper we present a new learning framework, called schema-based learning (SBL). SBL emphasizes the importance of the current existent structure (schemas), that defines a functioning system, for the incremental and autonomous construction of ever more complex structure to achieve ever more complex levels of functioning. We may rephrase this statement into the language of Schema Theory (Arbib 1992, for a comprehensive review) as the learning of new schemas based on the stock of current schemas. SBL emphasizes a fundamental principle of organization called coherence maximization, that deals with the maximization of congruence between the results of an interaction (external or internal) and the expectations generated for that interaction. A central hypothesis consists of the existence of a hierarchy of predictive internal models (predictive schemas) all over the control center-brain-of the agent. Hence, we will include predictive models in the perceptual, sensorimotor, and motor components of the autonomous agent architecture. We will then show that predictive models are fundamental for structural learning. In particular we will show how a system can learn a new structural component (augment the overall network topology) after being lesioned in order to recover (or even improve) its original functionality. Learning after lesioning is a special case of structural learning but clearly shows that solutions cannot be known/hardwired a priori since it cannot be known, in advance, which substructure is going to break down.

Animals↗

PubMed1994

The analysis of fluorophore-labeled glycans by high-resolution polyacrylamide gel electrophoresis.

Many important biological processes are mediated by the carbohydrate moieties of glycoconjugates. Their structural determination presents considerable difficulties because of both their complex structures and the small quantities which are available from biological sources. In order to address some of the problems in this field a new method of glycan analysis has been developed in which saccharides having a reducing end group are labeled covalently with a fluorophore and the derivatives separated with high resolution by polyacrylamide gel electrophoresis. Fluorophores have been used that enable the electrophoretic separation of positional isomers, anomers, and epimers of a variety of saccharides. Mixtures of complex glycans obtained by enzymatic cleavage from glycoproteins yield electrofluorograms showing high-resolution oligosaccharide profiles. Techniques for obtaining saccharide sequence information from isolated oligosaccharides have been developed. Subpicomolar quantities of individual saccharides can be detected using an imaging system based on a cooled charge-coupled device. The method enables rapid, simultaneous, parallel analyses of multiple samples. The analytical and clinical applications and potential of the system are discussed.

Carbohydrate Sequence↗

PubMed1985

Dorsal-ventral differences in the glia limitans of the spinal cord: an ultrastructural study in developing normal and irradiated rats.

The dorsal and ventral surfaces of the lumbosacral spinal cord were examined in normal and irradiated postnatal rats. In normal rats between three and 13 days postnatal (DP), the glia limitans (GL) of the ventral surface was a more complex structure than the dorsal GL. This greater degree of complexity was manifested in a greater number of subpial astrocytes, a greater number of radial glial processes and a more advanced state in differentiation of its constituents. In rats irradiated at three DP and examined at 13 DP, the ventral GL remained intact and relatively unaffected by the radiation. In contrast, the dorsal GL was disrupted, and Schwann cells were seen within the dorsal funiculus. The ventral GL of the rat lumbosacral spinal cord is a more substantial structure than the dorsal GL during normal development. This factor alone may account for the integrity of the barrier properties of the ventral GL following radiation. However, our observations suggest that subpial astrocytes of the dorsal GL are more susceptible to radiation damage at three DP than the subpial astrocytes and radial glia of the ventral GL.

Animals↗

PubMed2005

[Mitochondria and their role in cell metabolism].

Mitochondria are subcellular organelles of the endosymbiotic origin. They are bounded by double membrane and contain their own DNA. Recent advance in 3D microscopy have contributed a better understanding of mitochondrial structure. Mitochondria are highly dynamic organelles with a very complex structure of the inner membrane. In cells, mitochondria create an interconnected reticulum. Beyond a fundamental role in energy production, they also play key roles in thermogenesis, maintenance of cellular redox potential, Ca2+ homeostasis, ROS production, cell signaling and cell death. Disturbances in mitochondrial metabolism are known to play a role not only in rare genetics disorders, but have also been implicated in many common diseases of aging. Conventional studies of mitochondrial metabolism are based on the isolation of intact organelles. Because of mitochondrial complex roles rises a need to assay mitochondrial functions in situ. The activity of respiration and oxidative phosphorylation in intact and permeabilized cells can be measured by using high resolution respirometry. We can estimate various mitochondrial functions in living cells by using fluorescent cation dyes.

Animals↗

PubMed2006

Oxidative DNA cleavage induced by an iron(III) flavonoid complex: synthesis, crystal structure and characterization of chlorobis(flavonolato)(methanol) iron(III) complex.

A flavonol iron(III) complex, [Fe(flavonolato)(2)Cl(MeOH)], has been prepared. The compound has been characterized by X-ray crystallography, spectroscopy, magnetism and electronic paramagnetic resonance (EPR) at X- and Q-band. The geometrical environment around the metal is best described as rhombic distorted octahedral. This distortion has also been inferred from the magnetic measurements and from the EPR spectra at different temperatures, E/D(rhombicity parameter) approximately 0.06. The DNA cleavage activity of the iron(III) complex with and without ascorbate/hydrogen peroxide is reported. Mechanisms of the oxidative cleavage have been proposed when DNA strand scission is performed both with and without ascorbate/hydrogen peroxide activation.

Crystallography, X-Ray↗

PubMed1996

Structure model of a complex between the factor for inversion stimulation (FIS) and DNA: modeling protein-DNA complexes with dyad symmetry and known protein structures.

A method is presented to predict overall conformations of protein-DNA complexes on the basis of the known three-dimensional structures of the proteins. The method is restricted to proteins with a common twofold symmetry axis, which show only minor conformational changes upon binding to DNA. The method uses a numerical finite difference solution of the linearized Poisson-Boltzmann equation and subsequent energy minimization cycles. Structural parameters-the rotation angle of the DNA relative to the protein around the common symmetry axis, the protein-DNA distance, and intermolecular hydrogen-bonding contacts-are presented for two test cases, DNA bound to CAP (catabolite gene activator protein) and to the Cro-repressor of bacteriophage 434. The DNA curvature in the starting model of the docking procedure was chosen as a smoothed approximation of the conformation found in the X-ray structures of these complexes. The method is further used to predict the unknown structure of the complex between the factor for inversion stimulation (FIS) and DNA, which is bent upon binding to FIS. In contrast to the test cases, the unknown curvature of the starting model is derived from a calibration of electrostatic precalculations for different proteins according to crystallographically observed DNA bending. The results of the modeling are in good accordance with the experimentally observed overall structure of protein-DNA complexes for the two test cases; for FIS, they correspond to several of the experimentally proposed protein-DNA contacts.

DNA↗

PubMed1989

Evaluation of the murine metrial gland for immunological function.

The metrial gland (MG) is a transient uterine structure associated with rodent pregnancy. The gland is a complex structure consisting of stromal and vascular elements, as well as a population of histologically distinctive, large, granulated metrial gland (GMG) cells. The functions of the MG and of the GMG cells, as well as their relationship to the success of pregnancy, are unknown. Based upon morphological and morphometric studies it has been proposed that the MG might be involved in the immunology of pregnancy and that GMG cells could be immunocompetent. Explant cultures of MG have therefore been evaluated for immunological function. Lytic activity against the NK sensitive target cell line YAC and mitogen responsiveness could not be detected. MG tissue and medium conditioned by overnight culture of MG tissue (MG-CM) suppressed the response of murine spleen cells to Con A. MG-CM also reduced the lytic activity of splenic NK cells against YAC target cells. However, uptake of [3H]thymidine was elevated when YAC cells were cultured in MG-CM. The response of embryonic and uterine cells to growth in MG-CM was complex. MG-CM had little effect on isotope incorporation by decidual cells recovered at 6.5 days or by embryonic cells recovered from 12.5 day embryos. However, thymidine incorporation was less in MG-CM than in control medium for 12.5 day placental cells, 6.5 day embryonic sac, 6.5 day ectoplacental cone and 3.5 day blastocysts. Cytotoxicity and cytostasis accounted for reduced uptake of isotope in cultures of 3.5 day blastocysts and 6.5 day embryonic tissues. Loss of viability could not be detected in any other assays. Both YAC cells and unstimulated splenocytes showed altered morphology and improved viability when cultured in MG-CM. This study suggests that the only immunological role the MG might have during normal pregnancy is that of non-specific intra-uterine suppression. Alternatively, differential regulation of cell proliferation might be a function of the MG, within the pregnant uterus. The latter mechanism could also account for the apparent observation of non-specific immunosuppression.

Animals↗

PubMed2006

Crystallographic snapshot of a productive glycosylasparaginase-substrate complex.

Glycosylasparaginase (GA) plays an important role in asparagine-linked glycoprotein degradation. A deficiency in the activity of human GA leads to a lysosomal storage disease named aspartylglycosaminuria. GA belongs to a superfamily of N-terminal nucleophile hydrolases that autoproteolytically generate their mature enzymes from inactive single chain protein precursors. The side-chain of the newly exposed N-terminal residue then acts as a nucleophile during substrate hydrolysis. By taking advantage of mutant enzyme of Flavobacterium meningosepticum GA with reduced enzymatic activity, we have obtained a crystallographic snapshot of a productive complex with its substrate (NAcGlc-Asn), at 2.0 A resolution. This complex structure provided us an excellent model for the Michaelis complex to examine the specific contacts critical for substrate binding and catalysis. Substrate binding induces a conformational change near the active site of GA. To initiate catalysis, the side-chain of the N-terminal Thr152 is polarized by the free alpha-amino group on the same residue, mediated by the side-chain hydroxyl group of Thr170. Cleavage of the amide bond is then accomplished by a nucleophilic attack at the carbonyl carbon of the amide linkage in the substrate, leading to the formation of an acyl-enzyme intermediate through a negatively charged tetrahedral transition state.

Amidohydrolases↗

PubMed2004

Dimeric novel HSP40 is incorporated into the radial spoke complex during the assembly process in flagella.

The radial spoke is a stable structural complex in the 9 + 2 axoneme for the control of flagellar motility. However, the spokes in Chlamydomonas mutant pf24 are heterogeneous and unstable, whereas several spoke proteins are reduced differentially. To elucidate the defective mechanism, we clone RSP16, a prominent spoke protein diminished in pf24 axonemes. Unexpectedly, RSP16 is a novel HSP40 member of the DnaJ superfamily that assists chaperones in various protein-folding-related processes. Importantly, RSP16 is uniquely excluded from the 12S spoke precursor complex that is packaged in the cell body and transported toward the flagellar tip to be converted into mature 20S axonemal spokes. Rather, RSP16, transported separately, joins the precursor complex in flagella. Furthermore, RSP16 molecules in vitro and in flagella form homodimers, a characteristic required for the cochaperone activity of HSP40. We postulate that the spoke HSP40 operates as a cochaperone to assist chaperone machinery at the flagellar tip to actively convert the smaller spoke precursor and itself into the mature stable complex; failure of the interaction between the spoke HSP40 and its target polypeptide results in heterogeneous unstable radial spokes in pf24.

Amino Acid Sequence↗

PubMed2005

Structural allostery and binding of the transferrin*receptor complex.

The structural allostery and binding interface for the human serum transferrin (Tf)*transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions (Cheng, Y., Zak, O., Aisen, P., Harrison, S. C. & Walz, T. (2004) Structure of the human transferrin receptor.transferrin complex. Cell 116, 565-576). The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.

Allosteric Regulation↗

PubMed2002

Analysis of the conducting airway system in the lung: a new method combining morphometry with mathematical modeling for airway classification.

Although the lung is structurally complex, it is suitable for morphometric analysis of the structural determinants of lung function in health and disease. Analysis of the organized branching airways has been problematic because of the need to identify and classify airways before structural characteristics of different-order branches can be determined. Airway casts have been used to identify relationships between branches, measure some structural features, and develop mathematical models that describe simply the relationships between generations. However, cast preparation destroys surrounding tissue, including the airway wall, thus precluding analysis of these structural elements. We describe a new approach using tissue sections which combines the classification of airways into Strahler order (SO) with tissue structural analysis. Lung-tissue sections are prepared, and outer (OD) and inner (ID) diameters are determined over a wide range of airways. The line equation relating log OD vs. SO is determined using measured values for SO1 (terminal bronchioles) and SO8 (first branch bronchi). Mean ODs can then be calculated for each of the other SO groups, and measurements can be classified. Calculations can be made for the mean number of branches and airway lengths (given the log linear relationship of these factors with SO and morphometrically determined volume densities for airway lumen), and for individual airway resistance and total resistances for each SO. For an example, mean data are presented for airways in the adult sheep (n = 13). The methodology presented allows identification of subtle alterations in airway structures which may be affecting selected orders of airways, with specific implications for changes in lung function.

Airway Resistance↗

PubMed1995

Imaging: focus on hepatocellular carcinoma and liver transplantation.

The choice of management for the patient with HCC hinges on precise localization and staging of the disease process. All the major imaging modalities are employed to achieve this end. US is frequently the initial means of detection of the lesion. Since percutaneous needling may lead to tumour dissemination, the temptation to proceed to imaging-guided biopsy should be resisted until a full evaluation has been completed and it is clear that neither curative surgery nor transplantation is a therapeutic option. CT scanning is considered to be superior to ultrasound both in detection and staging of the disease. A variety of modifications to the technique, including CT arterio-portography and lipiodol-CT, is used to obtain optimum results. The role of MRI has not yet been established but initial results suggest that this may be the optimum means of scanning the patient following percutaneous or intra-arterial therapy. Angiography is generally performed prior to resection and may be combined with the delivery of chemotherapeutic and embolic agents pre-operatively or as a definitive palliative procedure. Imaging-guided percutaneous alcohol is also a useful palliative measure where the lesion is small. In the majority of cases, resection is not feasible. In a selected few liver transplantation is an option. Imaging requirements of the potential liver transplant candidate depend on the nature of the underlying liver problem. A general assessment including a chest X-ray and US with Doppler imaging of the hepatic vascular structures is sufficient in the majority. In children with complex structural anomalies and in patients with bile duct disease or tumours, the full range of investigations is required. US, cholangiography CT and angiography may all be required in the diagnosis and management of post-transplant complications.

Carcinoma, Hepatocellular↗

PubMed2006

Floral displays: genetic control of grass inflorescences.

Inflorescences in angiosperms are complex structures that have many different types of meristems. Among complex inflorescences, the best studied are in the grass family. Multiple inflorescence genes have been cloned from grasses over the past few years, many of them by positional cloning using the rice genome as a source of positional information. Several genes affect the apical meristem of the inflorescence differently from the lateral branch meristems, allowing morphological differentiation that permits diversification. ramosa1 (ra1), ra2, and ra3 have been cloned from maize and form part of a network of genes that control the production of lateral branching. Curiously, only ra2 is widely conserved; to date, ra1 and ra3 have been found only in Andropogoneae. Additional domestication genes that affect the inflorescence have also been cloned from maize, rice, and wheat.

Crops, Agricultural↗

PubMed2005

Solid state (13)C NMR and FT-IR spectroscopy of the cocoon silk of two common spiders.

The structure of the silk from cocoons of two common spiders, Araneus diadematus (family Araneidae) and Achaearanea tepidariorum (family Theridiidae) was investigated by means of (13)C solid state NMR and FT-IR spectroscopies. The combined use of these two techniques allowed us to highlight differences in the two samples. The cocoon silk of Achaearanea tepidariorum is essentially constituted by helical and beta-sheet structures, whereas that of Araneus diadematus shows a more complex structure, containing also beta-strands and beta-turns. Moreover, the former silk is essentially crystalline while the latter contains more mobile domains. The structural differences of the two cocoon silks are ascribed to the different habitat of the two species.

Animals↗

PubMed2005

Dissecting the roles of a strictly conserved tyrosine in substrate recognition and catalysis by pseudouridine 55 synthase.

Sequence alignment of the TruA, TruB, RsuA, and RluA families of pseudouridine synthases (PsiS) identifies a strictly conserved aspartic acid, which has been shown to be the critical nucleophile for the PsiS-catalyzed formation of pseudouridine (Psi). However, superposition of the representative structures from these four families of enzymes identifies two additional amino acids, a lysine or an arginine (K/R) and a tyrosine (Y), from a K/RxY motif that are structurally conserved in the active site. We have created a series of Thermotoga maritima and Escherichia coli pseudouridine 55 synthase (Psi55S) mutants in which the conserved Y is mutated to other amino acids. A new crystal structure of the T. maritima Psi55S Y67F mutant in complex with a 5FU-RNA at 2.4 A resolution revealed formation of 5-fluoro-6-hydroxypseudouridine (5FhPsi), the same product previously seen in wild-type Psi55S-5FU-RNA complex structures. HPLC analysis confirmed efficient formation of 5FhPsi by both Psi55S Y67F and Y67L mutants but to a much lesser extent by the Y67A mutant when 5FU-RNA substrate was used. However, both HPLC analysis and a tritium release assay indicated that these mutants had no detectable enzymatic activity when the natural RNA substrate was used. The combined structural and mutational studies lead us to propose that the side chain of the conserved tyrosine in these four families of PsiS plays a dual role within the active site, maintaining the structural integrity of the active site through its hydrophobic phenyl ring and acting as a general base through its OH group for the proton abstraction required in the last step of PsiS-catalyzed formation of Psi.

Binding Sites↗

PubMed2001

A comparison of the influences of alkoxide and thiolate ligands on the electronic structure and reactivity of molybdenum(3+) and tungsten(3+) complexes. preparation and structures of M(2)(O(T)Bu)(2)(S(t)Bu)(4), [Mo(S(t)Bu)(3)(NO)](2), and W(S(t)Bu)(3)(NO)(py).

M(2)(O(t)Bu)(6) compounds (M = Mo, W) react in hydrocarbon solvents with an excess of (t)BuSH to give M(2)(O(t)Bu)(2)(S(t)Bu)(4), red, air- and temperature-sensitive compounds. (1)H NMR studies reveal the equilibrium M(2)(O(t)Bu)(6) + 4(t)BuSH <==> M(2)(O(t)Bu)(2)(S(t)Bu)(4) + 4(t)BuOH proceeds to the right slowly at 22 degrees C. The intermediates M(2)(O(t)Bu)(4)(S(t)Bu)(2), M(2)(O(t)Bu)(3)(S(t)Bu)(3), and M(2)(O(t)Bu)(5)(S(t)Bu) have been detected. The equilibrium constants show the M-O(t)Bu bonds to be enthalpically favored over the M-S(t)Bu bonds. In contrast to the M(2)(O(t)Bu)(6) compounds, M(2)(O(t)Bu)(2)(S(t)Bu)(4) compounds are inert with respect to the addition of CO, CO(2), ethyne, (t)BuC triple bond CH, MeC triple bond N, and PhC triple bond N. Addition of an excess of (t)BuSH to a hydrocarbon solution of W(2)(O(t)Bu)(6)(mu-CO) leads to the rapid expulsion of CO and subsequent formation of W(2)(O(t)Bu)(2)(S(t)Bu)(4). Addition of an excess of (t)BuSH to hydrocarbon solutions of [Mo(O(t)Bu)(3)(NO)](2) and W(O(t)Bu)(3)(NO)(py) gives the structurally related compounds [Mo(S(t)Bu)(3)(NO)](2) and W(S(t)Bu)(3)(NO)(py), with linear M-N-O moieties and five-coordinate metal atoms. The values of nu(NO) are higher in the related thiolate compounds than in their alkoxide counterparts. The bonding in the model compounds M(2)(EH)(6), M(2)(OH)(2)(EH)(4), (HE)(3)M triple bond CMe, and W(EH)(3)(NO)(NH(3)) and the fragments M(EH)(3), where M = Mo or W and E = O or S, has been examined by DFT B3LYP calculations employing various basis sets including polarization functions for O and S and two different core potentials, LANL2 and relativistic CEP. BLYP calculations were done with ZORA relativistic terms using ADF 2000. The calculations, irrespective of the method used, indicate that the M-O bonds are more ionic than the M-S bonds and that E ppi to M dpi bonding is more important for E = O. The latter raises the M-M pi orbital energies by ca. 1 eV for M(2)(OH)(6) relative to M(2)(SH)(6). For M(EH)(3) fragments, the metal d(xz)(),d(yz)() orbitals are destabilized by OH ppi bonding, and in W(EH)(3)(NO)(NH(3)) the O ppi to M dpi donation enhances W dpi to NO pi* back-bonding. Estimates of the bond strengths for the M triple bond M in M(2)(EH)(6) compounds and M triple bond C in (EH)(3)M triple bond CMe have been obtained. The stronger pi donation of the alkoxide ligands is proposed to enhance back-bonding to the pi* orbitals of alkynes and nitriles and facilitate their reductive cleavage, a reaction that is not observed for their thiolate counterpart.

Journal Article↗

PubMed2005

Unsteady motion of receding contact lines of surfactant solutions: the role of surfactant re-self-assembly.

Re-self-assembly of surfactant molecules must occur at moving contact lines of soluble surfactant solutions. Molecules are transported into and out of the contact line region from four sources: the three interfaces meeting at the contact line and the fluid confined between the solid-liquid and liquid-vapor interfaces. As molecules move among these sources at the contact line, they must rearrange. The dynamics of this re-self-assembly has been shown to have a dominating effect on the structure of advancing contact lines, causing unsteady motion and complex structure of the contact line. It might be assumed that the re-self-assembly for receding contact lines leads to more steady contact line movement. However, in this article we show that for a wide variety of systems this is not true. Quasi-static distortions of the contact line occur as it retreats because of the inability of the surfactant to completely re-self-assemble at localized positions along the contact line.

Journal Article↗