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At least 253 records · Page 14Linked to original sources
PubMed1990

The fine structural localization of endogenous and exogenous peroxidase activity in human bone marrow mast cells under pathological conditions.

We have examined the ultrastructural characteristics of peroxidase activity in human bone marrow mast cells. These studies were performed in three patients with systemic mast cell disease, and in another six patients showing bone marrow mast cell hyperplasia. Endogenous peroxidase activity was localized in the perinuclear cisternae and strands of endoplasmic reticulum, but never in the granules. We have also demonstrated the "in vivo" existence of exogenous peroxidase activity in two of the three cases of systemic mast cell disease. The peroxidase internalization involved its binding to the plasma membrane, followed by its incorporation into the cell by a general endocytic process comprising the uptake of dispersed peroxidase-positive material mainly by phagocytosis of granular structures containing peroxidase. The exogenous peroxidase appeared in non-membrane bound granules, vacuoles or aggregates, but we have never seen the enzyme linked to the mast cell granules.

Adult↗
PubMed1990

Fine structural localization of RNA in myeloma cells detected by the enzyme-gold method.

The ultrastructural localization of RNA in myeloma cells was studied by the RNase-gold method. Gold particles indicating the presence of RNA were observed in large numbers, particularly in the granular component of the nucleolus and periphery of the rough endoplasmic reticulum, but not in the Golgi area, mitochondria, intranuclear inclusion bodies, cytoplasmic inclusion bodies, dense bodies, or cisternae of the rough endoplasmic reticulum. In the nuclear chromatin and nucleolus, gold particles were more numerous as these structures were less mature. They were found in larger numbers also in the cytoplasm of immature cells. In plasma cells from patients with macroglobulinemia, gold particles were fewer than in myeloma cells of multiple myeloma, but there was no difference in their distribution pattern.

Adult↗
PubMed2006

Localized structures in a nonlinear wave equation stabilized by negative global feedback: one-dimensional and quasi-two-dimensional kinks.

We study the evolution of fronts in a nonlinear wave equation with global feedback. This equation generalizes the Klein-Gordon and sine-Gordon equations. Extending previous work, we describe the derivation of an equation governing the front motion, which is strongly nonlinear, and, for the two-dimensional case, generalizes the damped Born-Infeld equation. We study the motion of one- and two-dimensional fronts, finding a much richer dynamics than for the classical case (with no global feedback), leading in most cases to a localized solution; i.e., the stabilization of one phase inside the other. The nature of the localized solution depends on the strength of the global feedback as well as on other parameters of the model.

Journal Article↗
PubMed2003

Bone tissue incorporates in vitro gallium with a local structure similar to gallium-doped brushite.

During mineral growth in rat bone-marrow stromal cell cultures, gallium follows calcium pathways. The dominant phase of the cell culture mineral constitutes the poorly crystalline hydroxyapatite (HAP). This model system mimics bone mineralization in vivo. The structural characterization of the Ga environment was performed by X-ray absorption spectroscopy at the Ga K-edge. These data were compared with Ga-doped synthetic compounds (poorly crystalline hydroxyapatite, amorphous calcium phosphate and brushite) and with strontium-treated bone tissue, obtained from the same culture model. It was found that Sr(2+) substitutes for Ca(2+) in the HAP crystal lattice. In contrast, the replacement by Ga(3+) yielded a much more disordered local environment of the probe atom in all investigated cell culture samples. The coordination of Ga ions in the cell culture minerals was similar to that of Ga(3+), substituted for Ca(2+), in the Ga-doped synthetic brushite (Ga-DCPD). The Ga atoms in the Ga-DCPD were coordinated by four oxygen atoms (1.90 A) of the four phosphate groups and two oxygen atoms at 2.02 A. Interestingly, the local environment of Ga in the cell culture minerals was not dependent on the onset of Ga treatment, the Ga concentration in the medium or the age of the mineral. Thus, it was concluded that Ga ions were incorporated into the precursor phase to the HAP mineral. Substitution for Ca(2+ )with Ga(3+) distorted locally this brushite-like environment, which prevented the transformation of the initially deposited phase into the poorly crystalline HAP.

Animals↗
PubMed1993

Local structure due to an aromatic-amide interaction observed by 1H-nuclear magnetic resonance spectroscopy in peptides related to the N terminus of bovine pancreatic trypsin inhibitor.

A synthetic peptide corresponding to the 15 N-terminal residues of bovine pancreatic trypsin inhibitor, with serine replacing the two cysteine residues, has been characterized by 1H-nuclear magnetic resonance spectroscopy. This peptide has a very disordered conformation that is essentially the same when it is part of the analogue of the (30-51) one-disulphide intermediate in folding. This confirms the conclusion of a previous paper, that the (30-51) intermediate is partially folded, with the N-terminal segment disordered. Local elements of non-random conformation were observed in the peptide. Especially prominent was an apparently electrostatic interaction between the face of the aromatic ring of Tyr10 and the amide group of Gly12, which caused the latter to have a very anomalous chemical shift. A similar interaction was observed in shorter peptides, especially in tetrapeptides with the sequences Tyr/Phe-X-Gly-Y. The local nature of this interaction indicates that it should be a general feature in peptides and in unfolded proteins with such a sequence.

Amides↗
PubMed1994

Complete coding sequence, deduced primary structure, chromosomal localization, and structural analysis of murine aggrecan.

We have isolated and sequenced overlapping cDNA clones encoding the entire core protein of aggrecan (the large aggregating chondroitin sulfate/keratan sulfate proteoglycan of cartilage) from three chondrocyte cDNA libraries of BALB/c mice and localized the aggrecan gene in mouse chromosome 7. We determined 7386 bp of the cDNA sequence, including 132 and 854 nucleotides of 5' and 3' untranslated regions, respectively. The core protein precursor is 2132 amino acids long (M(r) 222,008), including a 19-residue secretory signal peptide. The overall amino acid sequence of the mouse aggrecan shows 91.6% identity to rat and 72.5% to human aggrecan. Comparison of the amino acid sequences of various domains and subdomain structures of mouse aggrecan to known sequences of other species and related proteins (versican, neurocan, link protein, and lymphocyte homing receptor CD44) revealed high levels of identity of the G1, G2, and G3 globular domains and relatively less conserved structures in the interglobular and glycosaminoglycan-attachment regions. Epidermal growth factor (EGF)-like module was detected in only a minor fraction of aggrecan clones, while the complement regulatory protein (CRP)-like domain was regularly expressed in all samples.

Aggrecans↗
PubMed2001

Domain structure, localization, and function of DNA polymerase eta, defective in xeroderma pigmentosum variant cells.

DNA polymerase eta carries out translesion synthesis past UV photoproducts and is deficient in xeroderma pigmentosum (XP) variants. We report that poleta is mostly localized uniformly in the nucleus but is associated with replication foci during S phase. Following treatment of cells with UV irradiation or carcinogens, it accumulates at replication foci stalled at DNA damage. The C-terminal third of poleta is not required for polymerase activity. However, the C-terminal 70 aa are needed for nuclear localization and a further 50 aa for relocalization into foci. Poleta truncations lacking these domains fail to correct the defects in XP-variant cells. Furthermore, we have identified mutations in two XP variant patients that leave the polymerase motifs intact but cause loss of the localization domains.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization and structural localization of the reovirus lambda 3 protein.

The putative reovirus RNA polymerase, protein lambda 3, was characterized using antiserum prepared against a TrpE-lambda 3 fusion protein synthesized in Escherichia coli. Immunofluorescence microscopy showed that lambda 3 accumulated in perinuclear inclusion bodies in reovirus-infected cells. Analysis of lambda 3 accumulation in infected cells indicates that, once synthesized, lambda 3 is quite stable throughout the course of infection. Anti-lambda 3 serum did not immunoprecipitate virions, core particles or iodinated surface proteins of either virions or cores. These results indicate that lambda 3 is located in the inner part of the core. Experiments involving urea denaturation of purified reovirus cores indicate that lambda 3 cannot be selectively removed from the core without total denaturation of the core structure. When the dsRNA genome was eliminated from the core, lambda 3 remained associated with the other viral proteins in the core. Thus, lambda 3 appears to be a stable, structural component of the reovirus core, not bound to genomic dsRNA or free in soluble form inside the core.

Animals↗
PubMed1996

The structural localization of galanin, and its function in modulating acetylcholine release in the olfactory bulb of adult rat.

The localization of galanin immunoreactivity was analyzed within the olfactory bulb of adult rats. Galanin-positive neurons were differentially distributed among the bulb layers. The density of stained neurons was highest in the glomerular and external plexiform layers. According to morphology, size, location and arrangement, a large proportion of galanin-immunoreactive neurons corresponds to external tufted cells and short-axon neurons in the superficial part of the external plexiform and glomerular layers. A smaller number were middle tufted cells and short-axons neurons while only a few short-axon neurons were labeled in the granule cell layer. Galanin-stained nerve fibers had different structures (thick fibers with or without varicosities, and thin fibers with or without varicosities). Among them were afferent immunoreactive nerve fibers entering the bulb through the olfactory nerve layer, but penetrating superficial layers. Correspondingly, a large number of galanin-positive axons (with or without varicosities) were observed in the olfactory nerve layer. A number of galanin-positive nerve fibers was also present in the glomerular and internal plexiform layers, while these fibers were scarce in the granule cell layer, their density was lowest in the external plexiform layer. These results suggest that galanin-positive axons present in the olfactory bulb originate from at least four different sources. From the periphery axon bundles enter the bulb together with olfactory nerve fibers from the rostral direction and with a fiber bundle from the ventral posterior surface, i.e. at the border between the olfactory tract and the main olfactory bulb along a large blood vessel. Central sources are local interneurons in the olfactory bulb and some extrabulbar brain regions. Double-labeling experiments combining acetylcholinesterase histochemistry with galanin immunocytochemistry did not show any co-localization of acetylcholinesterase and galanin in nerve cell perikarya or nerve fibers. Synthetic porcine galanin (1-29) promoted acetylcholine release in olfactory bulb tissue slices, suggesting that galanin can effectively modulate cholinergic transmission and perhaps other forms of neuronal transmission. It is concluded that galanin may be significantly involved in olfactory processing at cellular and synaptic levels.

Acetylcholine↗
PubMedDate not supplied

A new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides: structure, localization, and function.

Recently, we identified a novel hypothalamic neuropeptide with a C-terminal LPLRF-amide sequence in the quail brain. This avian neuropeptide was shown to inhibit gonadotropin release from the cultured anterior pituitary. This peptide is the first hypothalamic peptide that inhibited gonadotropin release reported in vertebrates. We, therefore, termed it gonadotropin-inhibitory hormone (GnIH). After this finding, we found that GnIH-related peptides were present in the brains of other vertebrates, such as mammals, amphibians, and fish. These GnIH-related peptides possessed a LPXRF-amide (X=L or Q) motif at their C-termini in all investigated animals. Mass spectrometric analyses combined with immunoaffinity chromatography were powerful techniques for the identification of mature endogenous LPXRF-amide peptides. The identified LPXRF-amide peptides were found to be localized in the hypothalamus and brainstem areas, and to regulate pituitary hormone release. Subsequently, cDNAs that encode LPXRF-amide peptides were characterized in vertebrate brains. In this review, we summarize the identification, localization, and function of a new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides in vertebrates. Recent studies on the receptors for LPXRF-amide peptides will also be reviewed.

Amides↗
PubMed2004

A two-dimensional infrared study of localization, structure, and dynamics of a dipeptide in membrane environment.

Using methods of time-resolved two-dimensional infrared (2D-IR) spectroscopy, we approach the problem of the structural characterization of small polypeptide systems in a membrane environment. The 2D-IR spectra recorded for a model dipeptide in different environments demonstrated a significant change in the homogeneous and inhomogeneous broadenings of the amide I resonances when the molecule inserts either into a surfactant or a phospholipid membrane. Besides the change in the diagonal features in the 2D-IR response, we observe both intramolecular and intermolecular crosspeaks between the carbonyls of the dipeptide and the phospholipid. Considering the character of the diagonal peaks and the presence of the crosspeaks, we discuss the localization of the dipeptide moieties in the membrane. Using both the anisotropy and relative intensity of the observed intramolecular crosspeaks between the two amide I modes, we provide observables that help to determine the phi/psi-dihedral angles for the backbone of the dipeptide. Time dependent studies revealed slower conformational fluctuations of the dipeptide backbone in a membrane as compared to that in an aqueous environment.

Dipeptides↗
PubMed2001

Structure, localization and potential role of a novel molluscan trypsin inhibitor in Lymnaea.

Eggs and egg masses of the freshwater gastropod mollusc Lymnaea provide a microenvironment for developing embryos. Secretions of the exocrine albumen gland of Lymnaea are packaged in the eggs of an egg mass before the eggs are laid externally. The perivitelline fluid that directly surrounds individual oocytes is the main source of nutrition for developing embryos. During early stages of development, the perivitelline fluid is initially internalized by pinocytosis and degraded by lysosomes; in later stages, the embryo ingests the fluid. We previously found that the albumen gland produces large amounts of Lymnaea epidermal growth factor. The albumen gland also appears to produce significant amounts of a novel Lymnaea trypsin inhibitor (LTI), a second peptide that was purified and characterized from Lymnaea albumen gland extracts. The primary structure was determined by microsequence analysis, mass spectrometry, and C-terminal sequence analysis, and showed that LTI is a 57-residue glycosylated peptide. Comparison of the LTI sequence with other known serine protease inhibitors indicates that LTI is a member of the bovine pancreatic trypsin inhibitor family. Reverse phase-high performance liquid chromatography, microsequence analysis, mass spectrometry, and immunocytochemistry demonstrated that abundant amounts of intact LTI are packaged in egg masses. The presence of a trypsin inhibitor in the perivitelline fluid compartment of the egg mass may minimize digestion of peptides and proteins in the perivitelline fluid that are important for the development of the embryo, for example, Lymnaea epidermal growth factor.

Alkylation↗
PubMed1994

Local structural differences between alpha- and beta-elicitins shown by circular dichroism and ultraviolet difference spectroscopy.

In order to investigate differences in the conformation of elicitins exhibiting different levels of activity (toxicity to tobacco plants), the environment of the tyrosyl residues in four elicitins has been compared by different spectroscopic methods (difference absorption and circular dichroism). We compared two alpha-elicitins (capsicein and parasiticein) and two beta-elicitins (beta-cryptogein and beta-cinnamomin), that are 50-100 times more toxic than the alpha-ones. Thermal difference UV spectroscopy and titration experiments clearly showed the exposure of Tyr-85 by comparison of parasiticein lacking Tyr-85 and the accessibility of its hydroxyl group to the solvent. The adjacent Tyr-87 was also suggested to be located at the surface. In beta-cryptogein, beta-cinnamomin and capsicein the pK was measured at between 10.5 and 10.8, while in parasiticein it is higher (11.5) owing to a difference in the local environment. Thermal difference UV spectroscopy showed one more exposed tyrosine in beta-elicitins than in alpha-ones. This difference was attributed to Tyr-12, considering the more hydrophilic characteristic of the sequence around residue 13 in beta-elicitins and the role of this region in the toxicity. However, no difference in titration behaviour was noted among elicitins concerning Tyr-12. The other two tyrosines also presented an abnormal pK of titration (> 12). In all elicitins Tyr-47 was probably exposed, while Tyr-33 was probably buried and not titrated, except in beta-cinnamomin at very alkaline pH.

Algal Proteins↗
PubMed1980

Monoclonal antibodies used to probe acetylcholine receptor structure: localization of the main immunogenic region and detection of similarities between subunits.

Seventeen cell lines producing monoclonal antibodies against Torpedo californica (torpedo) acetylcholine receptor (AcChoR) and its subunits were established. By using these antibodies as probes, we identified: (i) a similar antigenic determinant on alpha and beta torpedo subunits, (ii) a similar antigenic determinant on gamma and delta subunits, (iii) antigenic determinants unique for alpha or beta torpedo AcChoR subunits, (iv) a small region on the alpha subunit that dominates the immunogenicity of native torpedo AcChoR in rats (a monoclonal antibody directed at this region could bind to rat AcChoR in vivo and cause passive experimental autoimmune myasthenia gravis), and (v) antigenic determinants on torpedo subunits recognized in AcChoR from other species. The unexpected similarities between alpha and beta and between gamma and delta subunits raise the possibility that the complex four-subunit structure of AcChoR was derived from a simpler precursor and suggests that these antigenic similarities might reflect some structural and functional homologies.

Animals↗
PubMed2006

Recurrent use of evolutionary importance for functional annotation of proteins based on local structural similarity.

The annotation of protein function has not kept pace with the exponential growth of raw sequence and structure data. An emerging solution to this problem is to identify 3D motifs or templates in protein structures that are necessary and sufficient determinants of function. Here, we demonstrate the recurrent use of evolutionary trace information to construct such 3D templates for enzymes, search for them in other structures, and distinguish true from spurious matches. Serine protease templates built from evolutionarily important residues distinguish between proteases and other proteins nearly as well as the classic Ser-His-Asp catalytic triad. In 53 enzymes spanning 33 distinct functions, an automated pipeline identifies functionally related proteins with an average positive predictive power of 62%, including correct matches to proteins with the same function but with low sequence identity (the average identity for some templates is only 17%). Although these template building, searching, and match classification strategies are not yet optimized, their sequential implementation demonstrates a functional annotation pipeline which does not require experimental information, but only local molecular mimicry among a small number of evolutionarily important residues.

Algorithms↗
PubMed1986

Cryptosporidium: cellular localization, structural analysis of absorptive cell-parasite membrane-membrane interactions in guinea pigs, and suggestion of protozoan transport by M cells.

In ilea of spontaneously infested guinea pigs, we examined the interface between the plasma membranes of cryptosporidia and absorptive cells using thin section and freeze fracture techniques. Initially, cryptosporidia invaginate microvilli, and the resulting redundant folds of membrane envelop the protozoan, thereby internalizing it in a membrane sac of host cell origin. Subsequently, a pentalaminar membrane fusion site develops at the base of the protozoan between the parasite's outer plasma membrane and the internalized host membrane. The membrane domains isolated by this fusion site are then modified: the host membrane disintegrates, and the isolated parasite membrane, which now directly contacts absorptive cell cytoplasm, becomes amplified. While cryptosporidia are restricted to the apex of absorptive cells, they may be found deep within the cytoplasm of M cells overlying Peyer's patches. Moreover, both intact and partially digested cryptosporidial organisms associate with macrophages subjacent to such M cells. These findings define the intracellular localization of cryptosporidia and suggest that cryptosporidial antigens may be sampled by intestinal lymphoid cells at sites underlying M cells.

Animals↗
PubMed1987

Structural localization of the O2-evolving apparatus to multimeric (tetrameric) particles on the lumenal surface of freeze-etched photosynthetic membranes.

Isolated appressed chloroplast membranes, highly enriched in photosystem II (PSII) activity, were examined by freeze-etch electron microscopy. The exposed surfaces of these Triton X-100 solubilized membrane fragments correspond to the lumenal or ESs surface of intact stacked thylakoid membrane regions (Dunahay, T. G., L. A. Staehelin, M. Seibert, P. D. Ogilvie, and S. P. Berg. 1984. Biochim. Biophys. Acta. 764:179-193). The sequential removal from this sample of three extrinsic proteins (17, 23, and 33 kD) associated with the O2-evolving apparatus and the concomitant loss of O2 evolution, was related to subtle changes in the height and substructure of characteristic multimeric (often tetrameric) particles that protrude from the ESs membrane surface. After removal of these proteins, the multimeric particles disappeared and dimeric particles of similar diameter but of lesser height (6.1 vs. 8.2 nm in the controls) were observed. Reconstitution of the depleted membrane fragments with the extrinsic proteins led to rebinding of the three proteins, to a 63% recovery of the control rates of O2 evolution, and to the reappearance of the larger multimeric particles. Analysis of the structural changes associated with the loss and rebinding of the extrinsic proteins is consistent with a stoichiometry of one PSII complex for either one or two copies of the 17-, 23-, and 33-kD proteins, and these are symmetrically arranged on the lumenal surface of the complex. These results demonstrate that the multimeric ESs particles correspond to part of the intact O2-evolving apparatus of PSII, thus confirming previous indirect studies relating these particles to PSII. The dimeric particles probably contain the rest of the O2-evolving complex.

Chlorophyll↗
PubMed1998

Partial characterization and three-dimensional-structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria.

The molecular basis for the metabolic defect in patients with phenylketonuria has been characterized for seven missense point mutations (R252G/Q, L255V/S, A259V/T and R270S) and a termination mutation (G272X) in an evolutionarily conserved motif of exon 7 in the catalytic domain of the human phenylalanine hydroxylase (hPAH) gene. The mutations were expressed in three heterologous in vitro systems. When expressed as fusion proteins with maltose-binding protein in Escherichia coli five of the mutant proteins demonstrated a defect in the normal ability of hPAH to fold and assemble as homotetramer/dimer, and they were mostly recovered as inactive aggregated forms. Only for the R252Q and L255V mutants were catalytically active tetramer and dimer recovered and for R252G some dimer, i.e. 20% (R252Q, tetramer), 44% (L255V, tetramer) and 4.4% (R252G, dimer) of the activity for the respective wild-type (wt) forms. When expressed by a coupled in vitro transcription-translation system, all the mutant enzymes were recovered as a mixture of non-phosphorylated and phosphorylated forms with a low homospecific activity (i.e. maximum 11% of wt-hPAH for the L255V mutant). When transiently expressed in human embryonic kidney (A293) cells a very low level of immunoreactive PAH protein was recovered in spite of normal PAH mRNA levels. All these mutations resulted in variant hPAH proteins which revealed a defect in oligomerization, an increased sensitivity to limited proteolysis in vitro, reduced cellular stability and a variable reduction in their catalytic activity. All these effects seem to result from structural perturbations of the monomer, and based on the crystal structure of the catalytic domain of hPAH, an explanation is provided for the impact of the mutations on the folding and oligomerization of the monomers.

Amino Acid Sequence↗