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PubMed1993

Morphogenesis in the fetal rat proximal colon: effects of cytochalasin D.

Two major morphogenetic events, epithelial conversion and fold formation, occur in the proximal rat colon during the last week of gestation. To evaluate the role of actin microfilaments in these two developmental processes, explants from the proximal colon of 19 day fetal rats were cultured in the presence of vehicle (0.1% dimethylsulfoxide), 0.1, 1.0, or 10 micrograms/ml of cytochalasin D (CD) for 24-48 hr. Explants as well as 19, 20, and 21 day in vivo controls were prepared for light, fluorescence, and electron microscopy. The distribution of actin filaments was determined by rhodamine-conjugated phalloidin binding and ultrastructural analysis of tissue fixed in the presence of tannic acid. Prior to fold formation, phalloidin binding was enhanced along the entire epithelial-mesenchymal interface. At the onset of fold formation, focal areas of intense fluorescence appeared at irregular intervals along the base of the stratified epithelium. Within 1 day, these focal intensities were localized at the apex of small forming folds. Additional changes occurring at the epithelial-mesenchymal interface in association with fold formation included: 1) ruffling of the previously smooth basal lamina, 2) a shape change in the subjacent mesenchymal cells from elongate to cuboidal along with the appearance of numerous processes abutting the basal lamina, and 3) a unique orientation of the associated collagen fibrils in some presumptive folds. Fold formation was inhibited in > 93% of explants cultured in the presence of 1.0 micrograms/ml CD. These explants appeared to be arrested precisely at the onset of fold formation. Epithelial conversion was also incomplete in these explants. These findings indicate an active role for actin in both fold formation and epithelial conversion.

Actins↗

PubMed2004

Arthroscopic biceps tenodesis using a bioabsorbable interference screw.

Arthroscopic biceps tenodesis has rarely been reported. The purpose of this study is to present our technique of arthroscopic biceps tenodesis using the Bio-Tenodesis (Arthrex, Naples, FL) screw system. This system uses a uniquely designed screwdriver that allows manipulation of the biceps tendon subacromially and intra-articularly, facilitates placement and maintenance of the biceps tendon at the base of the bone socket during interference screw insertion, and assures an adequate bone-tendon-screw interface.

Absorbable Implants↗

PubMed2001

Individually addressable gel-integrated voltammetric microelectrode array for high-resolution measurement of concentration profiles at interfaces.

The application of a novel voltammetric probe, based on an individually addressable gel-integrated microelectrode array (IA-GIME), for real-time, high-spatial resolution concentration profile measurements at interfaces is described. Reliability and validity of steep metal concentration gradients obtained with this novel system have been demonstrated by performing systematic tests at well-controlled liquid-liquid and liquid-solid interfaces. The liquid-liquid interface was formed by two layers of aqueous solutions with different components; only one layer contained trace metal ions (Pb(II) and Cd(II)); the individually addressable microelectrode array was placed at the interface of the liquid-liquid system; the concentration profiles were recorded as function of time; and the effective diffusion coefficients were calculated. The liquid-"solid" interface was formed from an aqueous solution layer overlying a bed of silica particles saturated with an aqueous solution. The sensor array has been used to monitor the diffusion processes of Tl(I) or Pb(II) from the liquid phase to the "solid" phase. The influences of porosity, geometry of the porous media, and complexation between metal ion and silica, on the diffusion processes, have been studied. All these results show that correct diffusion profiles of metal ions at interfaces can be obtained with 200-microm resolution with the IA-GIME. They also demonstrate that, for measurements in "solid" phase, the aforementioned factors must be considered carefully for correct calibration of any electrodes and the gel-integrated microelectrodes are unique tools to enable calibration of the sensors with synthetic solutions.

Journal Article↗

PubMed2006

Behavioral responses of epidermal Langerhans cells in situ to local pathological stimuli.

Pathological stimuli provoke coordinated changes in gene expression, surface phenotype, and function of dendritic cells (DCs), thereby facilitating the induction of adaptive immune responses. This concept of DC maturation was established mainly by studying epidermal Langerhans cells (LCs), a prototypic immature DC subset at the environmental interface. Taking advantage of I-Abeta-enhanced green fluorescent protein (EGFP) knock-in mice in which LCs can be visualized in intact skin, we recorded the dynamic movement of EGFP+ LCs by time-lapse confocal microscopy. LCs exhibited a unique behavior, termed dendrite surveillance extension and retraction cycling habitude (dSEARCH), characterized by rhythmic extension and retraction of dendrites through intercellular spaces between keratinocytes. When monitored after skin organ culture or subcutaneous injection of tumor necrosis factor alpha, LCs showed amplified dSEARCH and amoeba-like lateral migration between keratinocytes. Intravital imaging experiments further revealed steady-state dSEARCH motion in 5-10% of LCs. Topical application of a reactive hapten, DNFB, augmented dSEARCH and triggered lateral migration of LC in vivo. These observations introduce a new concept that in situ maturation of LCs is further accompanied by coordinated reprogramming of motile activities.

Animals↗

PubMed2001

The structure of the chloroplast F1-ATPase at 3.2 A resolution.

The structure of the F(1)-ATPase from spinach chloroplasts was determined to 3.2 A resolution by molecular replacement based on the homologous structure of the bovine mitochondrial enzyme. The crystallized complex contains four different subunits in a stoichiometry of alpha(3)beta(3)gammaepsilon. Subunit delta was removed before crystallization to improve the diffraction of the crystals. The overall structure of the noncatalytic alpha-subunits and the catalytic beta-subunits is highly similar to those of the mitochondrial and thermophilic subunits. However, in the crystal structure of the chloroplast enzyme, all alpha- and beta-subunits adopt a closed conformation and appear to contain no bound adenine nucleotides. The superimposed crystallographic symmetry in the space group R32 impaired an exact tracing of the gamma- and epsilon-subunits in the complex. However, clear electron density was present at the core of the alpha(3)beta(3)-subcomplex, which probably represents the C-terminal domain of the gamma-subunit. The structure of the spinach chloroplast F(1) has a potential binding site for the phytotoxin, tentoxin, at the alphabeta-interface near betaAsp(83) and an insertion from betaGly(56)-Asn(60) in the N-terminal beta-barrel domain probably increases the thermal stability of the complex. The structure probably represents an inactive latent state of the ATPase, which is unique to chloroplast and cyanobacterial enzymes.

Animals↗

PubMed2004

Structure and assembly of the RNA binding domain of bluetongue virus non-structural protein 2.

Bluetongue virus non-structural protein 2 belongs to a class of highly conserved proteins found in orbiviruses of the Reoviridae family. Non-structural protein 2 forms large multimeric complexes and localizes to cytoplasmic inclusions in infected cells. It is able to bind single-stranded RNA non-specifically, and it has been suggested that the protein is involved in the selection and condensation of the Bluetongue virus RNA segments prior to genome encapsidation. We have determined the x-ray structure of the N-terminal domain (sufficient for the RNA binding ability of non-structural protein 2) to 2.4 A resolution using anomalous scattering methods. Crystals of this apparently insoluble domain were obtained by in situ proteolysis of a soluble construct. The asymmetric unit shows two monomers related by non-crystallographic symmetry, with each monomer folded as a beta sandwich with a unique topology. The crystal structure reveals extensive monomer-monomer interactions, which explain the ability of the protein to self-assemble into large homomultimeric complexes. Of the entire surface area of the monomer, one-third is used to create the interfaces of the curved multimeric assembly observed in the x-ray structure. The structure reported here shows how the N-terminal domain would be able to bind single-stranded RNA non-specifically protecting the bound regions in a heterogeneous multimeric but not polymeric complex.

Amino Acid Sequence↗

PubMed1986

Data structures for DNA sequence manipulation.

Two data structures designated Fragment and Construct are described. The Fragment data structure defines a continuous nucleic acid sequence from a unique genetic origin. The Construct defines a continuous sequence composed of sequences from multiple genetic origins. These data structures are manipulated by a set of software tools to simulate the construction of mosaic recombinant DNA molecules. They are also used as an interface between sequence data banks and analytical programs.

Base Sequence↗

PubMed1994

Growth and differentiation properties of normal and transformed human keratinocytes in organotypic culture.

The growth and differentiation of human normal keratinocytes and their transformed counterparts were examined in organotypic cultures in which the keratinocytes were grown at the air-liquid interface on top of contracted collagen gel containing fibroblasts. We developed a modified culture procedure including the use of a mixed medium for keratinocytes and fibroblasts. Normal keratinocytes formed a three-dimensional structure of epithelium that closely resembled the epidermis in vivo, consisting of basal, spinous, granular and cornified layers. Cells synthesizing DNA were located in the lowest basal layer facing the collagen gel. Expressions of proteins involved in epidermal differentiation were examined by immunohistochemical staining and compared with those in skin in vivo. In the organotypic culture, transglutaminase, involucrin and filaggrin were expressed, as in the epidermis in vitro, most prominently in the granular layer. Type IV collagen, a component of basement membrane, was expressed at the interface between the keratinocyte sheet and the contracted collagen gel. Keratinocytes transformed by simian virus 40 or human papilloma virus (HPV) exhibited a highly disorganized pattern of squamous differentiation. In particular, HPV-transformed cells invaded the collagen gel. Organotypic culture is unique in that regulatory mechanisms of growth and differentiation of keratinocytes can be investigated under conditions mimicking those in vivo.

Antigens, Differentiation↗

PubMed2002

Y-chromosome analysis in Egypt suggests a genetic regional continuity in Northeastern Africa.

The geographic location of Egypt, at the interface between North Africa, the Middle East, and southern Europe, prompted us to investigate the genetic diversity of this population and its relationship with neighboring populations. To assess the extent to which the modern Egyptian population reflects this intermediate geographic position, ten Unique Event Polymorphisms (UEPs), mapping to the nonrecombining portion of the Y chromosome, have been typed in 164 Y chromosomes from three North African populations. The analysis of these binary markers, which define 11 Y-chromosome lineages, were used to determine the haplogroup frequencies in Egyptians, Moroccan Arabs, and Moroccan Berbers and thereby define the Y-chromosome background in these regions. Pairwise comparisons with a set of 15 different populations from neighboring European, North African, and Middle Eastern populations and geographic analysis showed the absence of any significant genetic barrier in the eastern part of the Mediterranean area, suggesting that genetic variation and gene flow in this area follow the "isolation-by-distance" model. These results are in sharp contrast with the observation of a strong north-south genetic barrier in the western Mediterranean basin, defined by the Gibraltar Strait. Thus, the Y-chromosome gene pool in the modern Egyptian population reflects a mixture of European, Middle Eastern, and African characteristics, highlighting the importance of ancient and recent migration waves, followed by gene flow, in the region.

Arabs↗

PubMed1991

Intravascular ultrasound imaging: in vivo peripheral and coronary artery studies.

Recent in vitro studies have demonstrated that intravascular ultrasound can obtain high-resolution cross-sectional images of arterial vessels. To further expand the use of this technique for in vivo visualization of peripheral and coronary vessels, we imaged 24 femoral and 13 carotid arteries from 19 sheep. Using a manual rotation technique, high-resolution images were obtained in 95% of the vessel sites with a rigid probe and in 82% of the vessel sites with a flexible catheter. In 14 of these arteries, good correlation was found between the lumen diameter measured by ultrasound and by angiography (P less than .001, r = .91). In addition, 6 left circumflex coronary arteries were imaged from 6 additional sheep by motor-driven rotation of the ultrasound probe at 1,800 rotations per minute, obtaining clear delineation of coronary lumen morphology and lumen-intima interface. Strong correlation was found also between intravascular ultrasound and cineangiography for coronary artery diameter measurement (P less than .001, r = .96). These studies demonstrate that this technique can provide high-resolution images of arterial vessels in vivo and may have unique advantages in diagnosis of atherosclerotic vascular disease and in the guidance of new catheter-based therapeutic modalities.

Animals↗

PubMed2005

Organic azides: an exploding diversity of a unique class of compounds.

Since the discovery of organic azides by Peter Griess more than 140 years ago, numerous syntheses of these energy-rich molecules have been developed. In more recent times in particular, completely new perspectives have been developed for their use in peptide chemistry, combinatorial chemistry, and heterocyclic synthesis. Organic azides have assumed an important position at the interface between chemistry, biology, medicine, and materials science. In this Review, the fundamental characteristics of azide chemistry and current developments are presented. The focus will be placed on cycloadditions (Huisgen reaction), aza ylide chemistry, and the synthesis of heterocycles. Further reactions such as the aza-Wittig reaction, the Sundberg rearrangement, the Staudinger ligation, the Boyer and Boyer-Aubé rearrangements, the Curtius rearrangement, the Schmidt rearrangement, and the Hemetsberger rearrangement bear witness to the versatility of modern azide chemistry.

Journal Article↗

PubMed1988

Acute lymphoblastic leukemia with a unique rearrangement between chromosomes 4 and 11.

A case of pre-B cell acute lymphoblastic leukemia (pre-B ALL) with a dir ins(11;4)(q23;q21q31) chromosome rearrangement is presented. The patient's clinical findings and history were similar to those described for the t(4;11)(q21;q23) subgroup of childhood ALL. These findings suggest that the interfacing of the distal breakpoint at band 4q21 to the proximal breakpoint of band 11q23 represents the primary cytogenetic change observed in this subgroup of ALL.

B-Lymphocytes↗

PubMed2001

Crystal packing interaction that blocks crystallization of a site-specific DNA binding protein-DNA complex.

We present here three high-resolution crystal structures of complexes between the DNA-binding domain of the heat-shock transcription factor (HSF) and DNA oligomers. Although the DNA oligomers contain HSF's specific binding sequence, called a heat-shock element, the crystal structures do not contain the specific protein-DNA complex. In one crystal structure, the 10 base pair DNA oligomer is statically disordered. In the other two related structures, the 12 base pair DNA oligomers are in unique positions, but the protein-DNA contacts in these two crystals are not sequence specific. In all three structures, the DNA appears to act as a rigid, polyanion scaffold to support columns of proteins in a crystalline lattice. A robust crystal packing interface between protein monomers obscures the true DNA-binding surface, known from previous genetic and biochemical studies. By redesigning the protein to interfere with the crystal lattice contacts, we were able to obtain physiologically relevant crystals in a specific protein-DNA complex. Thus, a crystal-packing interface was able to prevent the weak, but physiological relevant interactions between a protein and DNA.

Binding Sites↗

PubMed2001

Embryogenomics: developmental biology meets genomics.

Fundamental questions in developmental biology are: what genes are expressed, where and when they are expressed, what is the level of expression and how are these programs changed by the functional and structural alteration of genes? These questions have been addressed by studying one gene at a time, but a new research field that handles many genes in parallel is emerging. The methodology is at the interface of large-scale genomics approaches and developmental biology. Genomics needs developmental biology because one of the goals of genomics--collection and analysis of all genes in an organism--cannot be completed without working on embryonic tissues in which many genes are uniquely expressed. However, developmental biology needs genomics--the high-throughput approaches of genomics generate information about genes and pathways that can give an integrated view of complex processes. This article discusses these new approaches and their applications to mammalian developmental biology.

Animals↗

PubMed2002

Structural basis of phospholipase A2 inhibition for the synthesis of prostaglandins by the plant alkaloid aristolochic acid from a 1.7 A crystal structure.

This is the first structural observation of a plant product showing high affinity for phospholipase A(2) and regulating the synthesis of arachidonic acid, an intermediate in the production of prostaglandins. The crystal structure of a complex formed between Vipera russelli phospholipase A(2) and a plant alkaloid aristolochic acid has been determined and refined to 1.7 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) in the form of an asymmetric dimer with one molecule of aristolochic acid bound to one of them specifically. The most significant differences introduced by asymmetric molecular association in the structures of two molecules pertain to the conformations of their calcium binding loops, beta-wings, and the C-terminal regions. These differences are associated with a unique conformational behavior of Trp(31). Trp(31) is located at the entrance of the characteristic hydrophobic channel which works as a passage to the active site residues in the enzyme. In the case of molecule A, Trp(31) is found at the interface of two molecules and it forms a number of hydrophobic interactions with the residues of molecule B. Consequently, it is pulled outwardly, leaving the mouth of the hydrophobic channel wide open. On the other hand, Trp(31) in molecule B is exposed to the surface and moves inwardly due to the polar environment on the molecular surface, thus narrowing the opening of the hydrophobic channel. As a result, the aristolochic acid is bound to molecule A only while the binding site of molecule B is empty. It is noteworthy that the most critical interactions in the binding of aristolochic acid are provided by its OH group which forms two hydrogen bonds, one each with His(48) and Asp(49).

Animals↗

PubMed2001

Light field propagation by metal micro- and nanostructures.

The ability to sustain plasmon oscillations gives rise to unique properties of metal nanostructures, which can be exploited for the controlled manipulation of light fields on the nanoscale. In this context we investigate electromagnetic coupling effects within lithographically produced ensembles of gold nanoparticles with a photon scanning tunnelling microscope. To provide an interface between these nano-optical devices and classical far-field optics, we investigate surface plasmon propagation on microstructured metal thin films.

Journal Article↗

PubMed2003

Spontaneous N incorporation onto a Si(100) surface.

Initial nitridation of the Si(100) surface is investigated using photoemission, ion-scattering and ab initio calculations. After dissociation of NO or NH3, nitrogen atoms are found to spontaneously form a stable, highly coordinated N[triple bond]Si(3) species even at room temperature. The majority of the N species is incorporated into the subsurface Si layers occupying an interstitial site, whose atomic structure and unique bonding mechanism is clarified through ab initio calculations. This unusual adsorption behavior elucidates the atomistic mechanism of initial silicon nitride formation on the surface and has important implication on the N-rich layer formation at the SiO(x)N(y)/Si interface.

Journal Article↗

PubMed2004

Protein-protein interactions and transcriptional antagonism between the subfamily of NGFI-B/Nur77 orphan nuclear receptors and glucocorticoid receptor.

Glucocorticoids (Gc) act through the glucocorticoid receptor (GR) to enhance or repress transcription of glucocorticoid-responsive genes depending on the promoter and cellular context. Repression of proopiomelanocortin (POMC) gene expression by Gc was proposed to use different mechanisms. We described the POMC promoter Nur response element (NurRE) as a target for Gc repression. NGFI-B (Nur77), an orphan nuclear receptor, and two related factors, Nurr1 and NOR1, bind the NurRE as homo- or heterodimers to enhance POMC gene expression in response to CRH. Gc antagonize CRH-stimulated as well as NGFI-B-dependent transcription. We now show that GR antagonizes NurRE-dependent transcription induced by all members of the Nur77 subfamily and that these nuclear receptors can all interact directly with GR. Transcriptional antagonism as well as direct protein-protein interaction between NGFI-B and GR take place primarily via their respective DNA binding domains, although DNA binding itself and the GR homodimerization interface are not involved. In vivo, GR and Nur factors can be coimmunoprecipitated whereas GR is recruited to the POMC promoter upon glucocorticoid action. Thus, our data suggest a mechanism for transrepression between two nuclear receptors, GR and NGFI-B, that is unique, although quite similar to that proposed for transrepression between GR and activator protein 1 (AP-1) or nuclear factor-kappaB (NFkappaB).

Amino Acid Sequence↗