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At least 1,351 records · Page 75Linked to original sources
PubMed1998

Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n. (CAG)n repeats from the myotonic dystrophy locus.

The mechanism of disease-associated trinucleotide repeat length variation may involve slippage of the triplet-containing strand at the replication fork, generating a slipped-strand DNA structure. We recently reported formation in vitro of slipped-strand DNA (S-DNA) structures when DNAs containing triplet repeat blocks of myotonic dystrophy or fragile X diseases were melted and allowed to reanneal to form duplexes. Here additional evidence is presented that is consistent with the existence of S-DNA structures. We demonstrate that S-DNA structures can form between two complementary strands containing equal numbers of repeats. In addition, we show that both the propensity for S-DNA formation and the structural complexity of S-DNAs formed increase with increasing repeat length. S-DNA structures were also analyzed by electron microscopy, confirming that the two strands are slipped out of register with respect to each other and confirming the structural polymorphism expected within long tracts of trinucleotide repeats. For (CTG)50.(CAG)50 two distinct populations of slipped structures have been identified: those involving </=10 repeats per slippage, which appear as bent/kinked DNA molecules, and those involving >10 repeats, which have multiple loops or hairpins indicative of complex alternative DNA secondary structures.

DNA↗
PubMed2004

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure.

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.

Animals↗
PubMed1988

Structural changes in retinal pigmented epithelium of Rivulus marmoratus Poey embryos during development.

In Rivulus marmoratus development of retinal pigmented epithelium (RPE) parallels that of retinal photoreceptors. Although structurally functional by mid-incubation the full complex structure is not achieved even when the yolk-sac is absorbed (3-days post-hatched). Melanogenesis is evident at 0.2 incubation with premelanosomes present up to three days after hatching. The distribution of junctional complexes, basal membrane foldings and coated-pits throughout development is noted. Myeloid bodies, already present at mid-incubation, appear initially as single lenticular-shaped structures which later may form whorls, or coalesce around oil droplets, glycogen clusters or phagosomes thereby giving rise to myeloid patterns characteristic of a mature RPE. The functional significance of these changes is discussed.

Animals↗
PubMed1999

Surface Structural Ion Adsorption Modeling of Competitive Binding of Oxyanions by Metal (Hydr)oxides.

Spectroscopy has provided a progressive flow of information concerning the binding mechanism(s) of ions and their surface-complex structure. An important challenge in surface complexation models (SCM) is to connect the molecular microscopic reality to macroscopic adsorption phenomena. This is important because SCM alone provide insufficient insight in the binding mechanisms, and moreover, it is a priori not obvious that SCM, which describe the pH dependent adsorption correctly in simple systems, will predict the ion interaction under multicomponent conditions. This study elucidates the primary factor controlling the adsorption process by analysing the adsorption and competition of PO4, AsO4, and SeO3. We show that the structure of the surface-complex acting in the dominant electrostatic field can be ascertained as the primary controlling adsorption factor. The surface species of arsenate are identical with those of phosphate and the adsorption behavior is very similar. On the basis of the selenite adsorption, we show that the commonly used 2pK models are incapable to incorporate in the adsorption modeling the correct bidentate binding mechanism found by spectroscopy. The use of the bidentate mechanism leads to a proton-oxyanion ratio and corresponding pH dependency that are too large. The inappropriate intrinsic charge attribution to the primary surface groups and the condensation of the inner sphere surface complex to a point charge are responsible for this behavior of commonly used 2pK models. Both key factors are differently defined in the charge distributed multi site complexation (CD-MUSIC) model and are based in this model on a surface structural approach. The CD-MUSIC model can successfully describe the macroscopic adsorption phenomena using the surface speciation and binding mechanisms as found by spectroscopy. The model is also able to predict the anion competition well. The charge distribution in the interface is in agreement with the observed structure of surface complexes. Copyright 1999 Academic Press.

Journal Article↗
PubMed1996

X-ray structure of the uncomplexed anti-tumor antibody BR96 and comparison with its antigen-bound form.

The X-ray structure of the uncomplexed human chimeric Fab' of the anti-tumor antibody BR96 has been determined at 2.6 A resolution. The structure has been compared with Lewis Y antigen-complexed structures of BR96 which were determined previously. The comparison reveals segmental motions and/or conformational rearrangements of three CDR loops (L1, L3, and H2), whereas CDR H3 does not undergo changes upon complexation despite its significant main-chain contacts to the carbohydrate antigen. In light of the uncomplexed chimeric Fab' structure reported here, the previously observed high mobility of the CL:CH1 domains of the complexed chimeric BR96 Fab is rationalized as a "swinging" motion approximately about the axis of the elbow bend.

Animals↗
PubMed2006

Theoretical study on the isomeric structures and the stability of silylenoid (Tsi)Cl2SiLi (Tsi = C(SiMe3)3).

The structures and isomerization of silylenoid (Tsi)Cl(2)SiLi (Tsi = C(SiMe(3))(3)) were studied by density functional theory (DFT) at the B3LYP/6-31G(d) level. Four equilibrium structures and three isomeric transition states were located. The three-membered ring and p-complex structures, 1 and 2, are the two most stable forms. Two other local minima, the sigma-complex 3 and tetrahedron structure 4, should rearrange to 1 with very low barriers, and then to the most stable isomer 2. To exploit further the stability of silylenoid (Tsi)Cl(2)SiLi, the insertion reactions of 2 and silylene (Tsi)ClSi into the HF molecule have been investigated at the B3LYP/6-31G(d) level, respectively. The results show that the insertion of 2 into HF is very similar to that of (Tsi)ClSi into HF, but the latter is more favorable. To probe the influence of the substituent Tsi on the stability of silylenoid (Tsi)Cl(2)SiLi, the isomers and insertion reaction of silylenoid CH(3)Cl(2)SiLi were investigated in a similar way of those with (Tsi)Cl(2)SiLi. The results indicate that silylenoid containing very bulky group Tsi exhibits unusual stability because of the severe steric hindrance produced by Tsi at the center to which it is attached.

Journal Article↗
PubMed2003

Optimization of gas chromatography using short glass capillary column with mass spectrometry for identification and evaluation of commercial heavy alkylbenzene structures.

Heavy alkylbenzene (HAB) is one of the by-products when producing linear alkylbenzene (LAB). Identification of the fine structures of HAB is difficult because of its complex structure and high boiling point. The gas chromatography/mass spectrometry (GC/MS) technique with a short glass capillary column is the best method applicable to analysis of high-boiling and thermally labile compounds. The column efficiency is evaluated on the basis of Van Deemter curves. The experimental results show that the reduction in the optimum column efficiency for a short glass capillary column occurs along with a reduction in effective theoretical plates. A second important result is that the shift in the value of the flow velocity is related to the optimum point in height equivalent theoretical plate (HETP). The flow velocity is higher than that of conventional gas chromatography(GC). The optimized conditions were used to analyse HAB produced through the hydrogen fluoride (HF) alkylation process. The results show acceptable relative separation of a product in an extended boiling point range. The optimized conditions were also used to analyse three other special samples supplied by BASF company's AlCl(3 )alkylation process and to approve the previous structures. The results also show that HAB from Iran Chemical Industries Investment Company (ICIIC) produced through LAB complex consists of an additional part and has more linearity than the other three samples of HAB obtained from BASF.

Journal Article↗
PubMed2003

The structure of DNA within cationic lipid/DNA complexes.

The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed.

Cations↗
PubMed1990

[Colonoscopy in the diagnosis and follow-up of colonic polyps].

Out of 5000 patients examined endoscopically colon polyps were detected in 64%. The polyps were characterized by: higher incidence in 70-79-year-olds, high percentage of tubular adenomas (79%), large sizes of polyps with complex structure and their prevalence in the left colon, signs of malignancy in 0.64% of the removed polyps. The chance to develop cancer from tubular adenoma was 0.28%, tubular-villous, 1.4%, villous 3%. Recurrent polyps following polypectomy arose in 65% of cases. Tumor relapses emerging mainly within the first postoperative year occurred in 14% of cases. In 4 patients with cancer its onset was registered at the site of the removed large adenomas of complex histological structure. Colonoscopy proved an adequate method of diagnosis and follow-up of colon polyps. Endoscopic polypectomy can be considered as a measure to prevent colon cancer.

Aged↗
PubMed1994

In organello assembly of respiratory-chain complex I: primary structure of the 14.8 kDa subunit of Neurospora crassa complex I.

A cDNA encoding the 14.8 kDa subunit of complex I from Neurospora crassa was cloned and sequenced. The deduced primary structure of this subunit reveals a predominantly hydrophilic protein containing no obvious membrane-spanning domain. In agreement with this characteristic, we have localized the 14.8 kDa subunit in the peripheral arm of the enzyme. The 14.8 kDa subunit was found to be conserved in mammalian complex I. The conservation of this subunit in such distantly related organisms suggests that the 14.8 kDa subunit is an important component of complex I. We have used an in organello system to study the biosynthetic pathway of this subunit. The 14.8 kDa polypeptide could be efficiently imported into isolated mitochondria. Furthermore, a fraction of the in-vitro-imported subunit was found to assemble in complex I. This is the first time that assembly in complex I of an in-vitro-synthesized subunit is demonstrated.

Amino Acid Sequence↗
PubMed1986

Quantitation of the relationship between aortic endothelial intercellular cleft morphology and permeability to albumin.

The aortic endothelial intercellular cleft (AEC) is a determinant of permeability to macromolecules. This study compared the surface density and frequency of AEC profile types between areas of rat aorta that were permeable/impermeable to albumin-bound Evans Blue dye (EBD). Five adult male Wistar rats were given saturating i.v. doses of EBD and 30 min later were perfusion, fixed and their aortae excised. Samples of impermeable (White) and permeable (Blue) areas were prepared for EM. Sections were coded randomly and all AEC profiles observed were assigned to 4 different morphological classes. Ten to 15 micrographs per sectioned sample, per area, per rat were taken to determine the surface density of AEC's relative to the endothelial cell layer. The frequency distribution of AEC profile types from Blue areas was significantly (P less than 0.01) different from that of the White areas. The Blue regions had relatively more AEC profiles of a less complex structure than did the White areas. The majority of cleft profiles in the Blue areas were of the simple 'overlap' type, whereas the commonest in the White areas were the 'mortise' type. The mean surface density of the AEC's in Blue areas was 0.066 +/- 0.002 micron-1, which was significantly (P less than 0.05) less than in White areas (0.11 +/- 0.002 micron-1). These findings confirm earlier qualitative observations and indicate that AEC's in areas permeable to macromolecules, such as albumin, are less complex in structure and of lower surface area than those in areas relatively impermeable to such macromolecules.

Albumins↗
PubMed1992

Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and cytochrome c.

The crystal structure of a 1:1 complex between yeast cytochrome c peroxidase and yeast iso-1-cytochrome c was determined at 2.3 A resolution. This structure reveals a possible electron transfer pathway unlike any previously proposed for this extensively studied redox pair. The shortest straight line between the two hemes closely follows the peroxidase backbone chain of residues Ala194, Ala193, Gly192, and finally Trp191, the indole ring of which is perpendicular to, and in van der Waals contact with, the peroxidase heme. The crystal structure at 2.8 A of a complex between yeast cytochrome c peroxidase and horse heart cytochrome c was also determined. Although crystals of the two complexes (one with cytochrome c from yeast and the other with cytochrome c from horse) grew under very different conditions and belong to different space groups, the two complex structures are closely similar, suggesting that cytochrome c interacts with its redox partners in a highly specific manner.

Amino Acid Sequence↗
PubMed1998

Glycerol kinase from Escherichia coli and an Ala65-->Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation.

BACKGROUND: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effectors with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers. GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle. Members of this family include actin, hexokinase and the heat shock protein hsc70. RESULTS: We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIAGlc-GK complex structure. The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 A translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIAGlc-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (IIe474-->Asp) in complex with IIAGlc; this complex crystallized isomorphously to the wild type IIAGlc-GK complex. Site-directed mutants of GK with substitutions at the IIAGlc-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity. CONCLUSIONS: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site. Mutational analysis and the structure of the IIAGlc-GK(IIe474-->Asp) complex suggest the conformational transition of the IIAGlc-binding site to be an essential aspect of IIAGlc regulation.

Alanine↗
PubMed1998

Virtual otoscopy.

Imaging techniques assist the surgeon in diagnosis of disease, surgical planning, and providing image guidance during surgery. Endoscopy has the drawback of being a minimally invasive procedure and limiting visualization to the inner surface of the lumen. Ultrasound, CT, and MR imaging show volumes of tissue beyond the lumen wall; however, their planar, two-dimensional representations require mental reconstruction of anatomic structures, which often proves difficult with the small, complex structures within the temporal bone. To improve three-dimensional visualization of the inner ear, we successfully completed a virtual model that can be displayed as a contiguous, three-dimensional luminal view, known as virtual otoscopy, which emulates traditional endoscopy. A concomitant global view and a view of the related CT slice adds a distinct advantage in the presentation and study of this complex organ. Advances in computer and software technology may overcome the time and cost factors that, at present, limit widespread use of virtual otoscopy. Overall, virtual otoscopy stands as a promising new visualization technique for elucidation of the middle ear, inner ear, and temporal bone structures.

Computer Simulation↗
PubMed1997

Not just another Fab: the crystal structure of a TcR-MHC-peptide complex.

The structure of a ternary complex formed between a T-cell receptor, a major histocompatibility complex (MHC) protein and a viral peptide provides new insights into the cellular immune response. The results provide a molecular basis for understanding the development of T cells and the reactions leading to transplant rejection and autoimmunity.

Gene Products, tax↗
PubMed2003

RNA polymerase II/TFIIF structure and conserved organization of the initiation complex.

The structure of an RNA polymerase II/general transcription factor TFIIF complex was determined by cryo-electron microscopy and single particle analysis. Density due to TFIIF was not concentrated in one area but rather was widely distributed across the surface of the polymerase. The largest subunit of TFIIF interacted with the dissociable Rpb4/Rpb7 polymerase subunit complex and with the mobile "clamp." The distribution of the second largest subunit of TFIIF was very similar to that previously reported for the sigma subunit in the bacterial RNA polymerase holoenzyme, consisting of a series of globular domains extending along the polymerase active site cleft. This result indicates that the second TFIIF subunit is a true structural homolog of the bacterial sigma factor and reveals an important similarity of the transcription initiation mechanism between bacteria and eukaryotes. The structure of the RNAPII/TFIIF complex suggests a model for the organization of a minimal transcription initiation complex.

Animals↗
PubMed1998

Reconstitution of core light-harvesting complexes of photosynthetic bacteria using chemically synthesized polypeptides. 2. Determination of structural features that stabilize complex formation and their implications for the structure of the subunit complex.

Chemically synthesized polypeptides have been utilized with a reconstitution assay to determine the role of specific amino acid side chains in stabilizing the core light-harvesting complex (LH1) of photosynthetic bacteria and its subunit complex. In the preceding paper [Meadows, K. A., Parkes-Loach, P. S., Kehoe, J. W., and Loach, P. A. (1998) Biochemistry 37, 3411-3417], it was demonstrated that 31-residue polypeptides (compared to 48 and 54 amino acids in the native polypeptides) having the same sequence as the core region of the beta-polypeptide of Rhodobacter sphaeroides (sph beta 31) or Rhodospirillum rubrum (rr beta 31) could form subunit-type complexes. However, neither polypeptide interacted with the native alpha-polypeptides to form a native LH1 complex. In this paper, it is demonstrated that larger segments of the native Rb. sphaeroides beta-polypeptide possess native behavior in LH1 formation. Polypeptides were synthesized that were six (sph beta 37) and ten amino acids (sph beta 41) longer than sph beta 31. Although sph beta 37 exhibited behavior nearly identical to that of sph beta 31, sph beta 41 behaved more like the native polypeptide. In the case of rr beta 31, a polypeptide with four additional amino acids toward the C terminus was synthesized (rr beta 35). Because LH1-forming behavior was not recovered with this longer polypeptide, one or more of the three remaining amino acids at the C-terminal end of the native beta-polypeptide seem to play an important role in LH1 stabilization in Rs. rubrum. Three analogues of the core region of the Rb. sphaeroides beta-polypeptide were synthesized, in each of which one highly conserved amino acid was changed. Evidence was obtained that the penultimate amino acid, a Trp residue, is especially important for subunit formation. When it was changed to Phe, the lambda Max of the subunit shifted from 823 to 811 nm and the association constant decreased about 500-fold. Changing each of two other amino acids had smaller effects on subunit formation. Changing Trp to Phe at the location six amino acid residues toward the C terminus from the His coordinated to Bchl resulted in an approximately 10-fold decrease in the association constant for subunit formation but did not affect the formation of a LH1-type complex compared to sph beta 31. Finally, changing Arg to Leu at the location seven amino acid residues toward the C terminus from the His coordinated to Bchl decreased the association constant for subunit formation by about 30-fold. In this case, no LH1-type complex could be formed. On the basis of these results, in comparison with the crystal structure of the LH2 beta-polypeptide of Rhodospirillum molischianum, two possible structures for the subunit complex are suggested.

Amino Acid Sequence↗
PubMed1999

Modelling of factor Xa-inhibitor complexes: a computational flexible docking approach.

In order to understand the structural basis of Factor Xa (FXa) specificity, structural complexes of FXa with its synthetic inhibitors are determined using a computational docking approach. The AutoDock suite of programs is used to determine the binding modes of the synthetic inhibitors such as 3- and 4-amidinobenzylphenyl ether (ABP), amidinophenyl pyruvic acid (APPA), diamidinobenzofuranyl ethene (DABE), and DX-9065a 2-(5'-amidino-2'-benzofuranyl)-3-(7'amidino-2'-napthyl)-propionic acid (ABAP) to FXa. The synthetic inhibitors docked in the present study are different in size, nature of linkage, and properties. Two sets of simulations were carried out for synthetic inhibitors docking to FXa. In the first set of simulations, no explicit water molecules were included. In the second set of simulations two explicit solvent molecules were considered. In all the computationally predicted synthetic inhibitor complexes of FXa, the specificity pocket residue Asp-189 is involved in hydrogen bonding with the bound inhibitor. The active site water molecule WAT522 is involved in hydrogen bonding with all the bound inhibitors. The computed energies clearly discriminate the high affinity from low affinity binders.

Amidines↗