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PubMed2004

Structural features of the botulinum neurotoxin molecule that govern binding and transcytosis across polarized human intestinal epithelial cells.

Experiments were done to help localize the minimum essential domain within the botulinum toxin molecule that is necessary for binding and transport across human gut epithelial cells. The data demonstrated that the neurotoxin alone, in the absence of auxiliary proteins, undergoes transcytosis. The neurotoxin by itself was examined in the single chain (unnicked serotype B) and dichain (nicked serotype B, nicked serotype A) forms, and all displayed the ability to bind and penetrate epithelial barriers. In addition, the single chain and dichain molecules were examined in the oxidized and reduced states, and again all forms were transported. To further define the minimum essential domain, experiments were done with two toxin fragments: 1) the heavy chain, which was derived from native toxin, and 2) the carboxy-terminal portion of the heavy chain, which was generated by recombinant techniques. Interestingly, both fragments were fully competent in crossing epithelial barriers. These data suggest that a polypeptide derived from the toxin could be used as a carrier domain to transport other molecules across epithelial cells. In related experiments, physiological (i.e., potassium depletion) and pharmacological (i.e., chlorpromazine) manipulations were used to implicate clathrin-coated pits/vesicles as the structures responsible for endocytosis of toxin.

Animals↗

PubMed1983

Structural features of a phased nucleosome core particle.

Chicken erythrocyte inner histones associate with a cloned 260-base-pair (bp) segment of Lytechinus variegatus DNA in a unique location. The fragment contains a 120-bp segment encoding 5S rRNA, a 90-bp flanking sequence to the 5' side of the transcribed segment, and a 50-bp downstream flanking sequence. Association of DNA, uniquely labeled at one end or the other and at either the 3' or the 5' terminus of a given strand, with histones at 0.1 M ionic strength leads to formation of a compact complex which sediments at about 13 S. Analysis of cutting of the complex by DNase I shows that protection from the nuclease is confined to a region beginning 20 bp from the left end of the segment and extending to about 165 bp from the left end. Within the protected region, the two DNA strands differ in their susceptibilities to the nuclease, the precise location of nuclease cutting sites and the spacing between these sites, and the relative susceptibilities of specific cutting locations. It seems that information present in DNA and the histone octomer is sufficient to create a precisely phased nucleosome in which interactions of the two DNA strands with histones are not the same. The structure of this unique nucleosome is not predicted by the intellectual model based on studies of mixed populations of nucleosome core particles.

Animals↗

PubMed1989

Structural features of Borrelia burgdorferi--the Lyme disease spirochete: silver staining for nucleic acids.

Borrelia burgdorferi--the Lyme disease spirochete--was grown in modified Kelly medium and characterized by transmission and by scanning electron microscopy. Using silver staining procedures which preferentially bind to nuclear components of eukaryotic cells, signal could be detected by backscattered electron imaging throughout the length of the prokaryotic spirochete. Interestingly, however, the highest levels of backscattered signal were observed in naturally elaborated membrane blebs that were visible attached to cell surfaces and free in the medium. These membrane vesicles could be enriched by filtration through nitrocellulose or Anopore membranes and by differential centrifugation. The possibility of contaminating cellular DNA coating the membrane vesicles was ruled out by exhaustive digestion with pancreatic DNAse I. Intact DNA was demonstrated both by lysing blebs directly on the surface of microscope grids and by extracting molecules from purified bleb preparation with detergents and solvents. Both linear and circular DNA molecules could be identified in purified membrane blebs. A simple, one-step, alternative silver staining procedure is described which appears to effectively label the protein-nucleic acid complexes contained in the membrane vesicles of the human pathogen B. burgdorferi, and may provide an important method to track and to define the biological function of these structures.

Borrelia burgdorferi↗

PubMed2004

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea.

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular "fungal pegs" within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.

Ericaceae↗

PubMed2004

In silico identification and structural features of six new genes similar to MATER specifically expressed in the oocyte.

In the present work, we have used the in silico subtraction methodology to identify six new mouse genes similar to NALP5/MATER, whose ESTs were represented almost exclusively in egg libraries. Five genes were selected for RT-PCR and/or in situ hybridization. These experiments confirmed their oocyte restricted expression. Five of these genes are localized on mouse chromosome 7, as is NALP5/MATER; among them, three are localized in a 300 kb cluster.

Amino Acid Sequence↗

PubMed2004

Structural features of nucleosomes reorganized by yeast FACT and its HMG box component, Nhp6.

The Saccharomyces cerevisiae Spt16/Cdc68, Pob3, and Nhp6 proteins (SPN or yFACT) bind to and alter nucleosomes in vitro, providing a potential explanation for their importance in both transcription and replication in vivo. We show that nucleosomes bound by either Nhp6 alone or the yFACT complex remain largely intact and immobile but are significantly reorganized, as indicated by changes in the pattern of sensitivity to DNase I and enhanced digestion by some restriction endonucleases. In contrast, yFACT enhanced access to exonuclease III only at very high levels of enzyme, suggesting that the DNA near the entry and exit sites of nucleosomes is largely unperturbed and that the position of the histone octamers relative to the DNA is not altered during reorganization. DNase I sensitivity was enhanced at sites clustered near the center of the nucleosomal DNA, away from the entry and exit points, and the pattern of nuclease sensitivity was only mildly affected by the configuration of linker extensions, further indicating that linkers play only a minor role in the reorganization of nucleosomes by yFACT. The DNA in contact with H2A-H2B dimers is therefore the region whose nuclease sensitivity was the least affected by yFACT reorganization. The most dramatic changes in nucleosome structure occurred when Spt16-Pob3 and the HMG box protein Nhp6 were both present, but Nhp6 alone altered DNase I sensitivity at some specific sites, supporting an independent role for this class of proteins in the general management of chromatin properties. yFACT activity does not require ATP hydrolysis and does not alter the position of nucleosomes, indicating that it acts through a mechanism distinct from chromatin remodeling. The results presented here suggest instead that yFACT promotes polymerase progression by reorganizing nucleosome cores into a less inhibitory conformation in which the properties of DNA sequences near the center of the nucleosomes are altered.

Animals↗

PubMed1987

Structural features required for ligand binding to the beta-adrenergic receptor.

On the basis of the homology between the amino acid sequences of the beta-adrenergic receptor (beta AR) and the opsin proteins we have proposed that the ligand binding domain lies within the seven transmembrane hydrophobic regions of the protein, which are connected by hydrophilic regions alternatively exposed extracellularly and intracellularly. We have systematically examined the importance of each of these regions by making a sequential series of deletions in the gene for the hamster beta AR which encompass most of the protein coding region. The ability of the corresponding mutant receptors to be expressed, localized to the cell membrane, and bind beta-adrenergic ligands has been analyzed, using transient expression in COS-7 cells. The hydrophobic regions and the hydrophilic segments immediately adjacent to the membrane cannot be removed without affecting the processing and membrane localization of the beta AR. However, most of the hydrophilic regions appear to be dispensable for ligand binding. In addition, we observed that substitution of the conserved cysteine residues at positions 106 and 184 dramatically altered the ligand binding characteristics of the beta AR, suggesting the occurrence of a disulfide bond between these two residues in the native protein. These data are discussed in terms of the tertiary structure of the beta AR.

Amino Acid Sequence↗

PubMed1999

Phospholipid-assisted refolding of an integral membrane protein. Minimum structural features for phosphatidylethanolamine to act as a molecular chaperone.

Escherichia coli-derived phosphatidylethanolamine (PE) or PE with fully saturated fatty acids was able to correct in vitro a defect in folding in the lipid-dependent epitope 4B1 of lactose permease (LacY) resulting from in vivo assembly in the absence of PE. PE plasmalogen, PE with two unsaturated fatty acids, and lyso-PE, which all do not favor bilayer organization, did not support proper refolding. Proper refolding occurred when these latter lipids were mixed with a bilayer-forming lipid (phosphatidylglycerol), which alone could not support refolding. L-Phosphatidylserine (PS; natural diastereomer) did support proper refolding. PE derivatives of increasing degrees of methylation were progressively less effective in supporting refolding, with phosphatidylcholine being completely ineffective. Therefore, the properties of nonmethylated aminophospholipids capable of organization into a bilayer configuration are essential for the recovery of the native state of epitope 4B1 after misassembly in vivo in the absence of PE. Neither D-PS (sn-glycero-1-phosphate backbone) nor P-D-S (D-serine in the head group) is competent in supporting proper refolding unless used in binary mixtures with phosphatidylglycerol. The detailed characterization of phospholipid-assisted refolding reported here further supports a specific rather than nonspecific role for PE in structural maturation of lactose permease in vivo (Bogdanov, M., and Dowhan, W. (1998) EMBO J. 17, 5255-5264).

Amines↗

PubMed2000

Regulation of FGF-1 mitogenic activity by heparan sulfate oligosaccharides is dependent on specific structural features: differential requirements for the modulation of FGF-1 and FGF-2.

The interaction of heparan sulfate (HS) (and the closely related molecule heparin) with FGF-1 is a requirement for enabling the growth factor to activate its cell surface tyrosine kinase receptor. However, little is known about the regulatory role of naturally occurring cell surface HS in FGF-1 activation. We have addressed this issue by utilizing a library of HS oligosaccharides, which are defined in both length and sulfate content. Mitogenic activation assays using these oligosaccharides showed that HS contained both FGF-1 activatory and inhibitory sugar sequences. Further analysis of these oligosaccharides showed a clear correlation between FGF-1 promoting activity and their 6-O-sulfate content. The results, in particular with the dodecasaccharide sequences, suggested that specific positioning of 6-O-sulfate groups may be required for the promotion of FGF-1 mitogenic activity. This may also be true for 2-O-sulfate groups though the evidence was not as conclusive. Differential activation of FGF-1 and FGF-2 was also observed and found to be mediated by both oligosaccharide length and sulfation pattern, with different specific O-sulfate positioning being implicated for the promotion of different growth factors. These results suggest that variation and tight control of the fine structure of HS may allow cells to not only control their positive/negative responses to individual FGFs but also to change specificity towards promotion of different members of the FGF family.

Animals↗

PubMed1993

Structural features of polysaccharides that induce intra-abdominal abscesses.

The capsular polysaccharide complex from Bacteroides fragilis promotes the formation of intra-abdominal abscesses--a pathologic host response to infecting microorganisms. This complex consists of two distinct polysaccharides, each with repeating units that have positively charged amino groups and negatively charged carboxyl or phosphate groups. Analysis of these polysaccharides as well as other charged carbohydrates before and after chemical modification revealed that these oppositely charged groups are required for the induction of intra-abdominal abscesses in a rat model.

Abdomen↗

PubMed1990

Anthracycline antibiotics with high liposome entrapment: structural features and biological activity.

We evaluated the entrapment of 21 different water-insoluble aglycones or anthracycline antibiotics in multilamellar liposomes composed of dimyristoyl phosphatidyl choline and dimyristoyl phosphatidyl glycerol at a 7:3 molar ratio. The drug:lipid weight ratio was 1:15 to 1:50. The different analogues tested were modified at position 4 in the aglycone portion (4-demethoxy) and/or positions 2' (halo), 3' (hydroxy, acetoxy), or 4' (epi, acetoxy) in the sugar portion. The entrapment efficiency was assessed by measuring the amount of free drug remaining in the supernatant after centrifugation of the liposomes and by direct examination of the pellets by fluorescent microscopy. Optimal entrapment (greater than 98%) was observed with only four compounds: 4-demethoxyadriamycinone; 2'-iododaunorubicin; 4-demethoxydaunorubicin; and 2'-iodo-3'-hydroxy-4'-epi-4-demethoxydoxorubicin (Compound 22). All other compounds showed significant drug precipitation outside the multilamellar vesicles when observed by fluorescent microscopy. Compound 22, entrapped in liposomes, was evaluated in vivo against i.p. L-1210 leukemia by the i.p. route, and liver metastases of M5076 reticulosarcoma by the i.v. route. In both models, liposome-entrapped Compound 22 was more active than doxorubicin at the optimal dose [median survival (given in percentage) of treated to control animals was for L-1210, greater than 600 versus 212; for M5076, 200 versus 133]. 4-Demethoxy and 2'-iodo are structural modifications that markedly enhance the affinity of anthracycline antibiotics for lipid bilayers without compromising biological activity. These findings will serve as a guideline to obtain liposome-anthracycline preparations, with optimal formulation characteristics, enhanced tumor-targeting properties, and non-cross-resistance with doxorubicin.

Animals↗

PubMedDate not supplied

[Structural features of pregna-D'-pentaranes determining their interaction with three rat proteins].

Competition of a number of progesterone 16alpha,17alpha-cycloalkane derivatives with 3H-labeled ligands for the binding sites of the rat uterine progesterone receptor, uterine pentaranophilin, and blood serum pentaranophilin was studied. We found that the selective ligands for the progesterone receptor are progesterone, 16alpha,17alpha-cyclopropanoprogesterone, and 16alpha,17alpha-cyclopent-3'-enoprogesterone and the selective ligands for serum pentaranophilin are 6alpha-methyl-16alpha,17alpha-cyclohexanopregna-1,4-diene-3,20-dione and 3beta-hydroxy-16alpha,17alpha-cyclohexanopregn-5-en-20-one. No selective ligands for the uterine pentaranophilin were found. The majority of substituents in rings A, B, and D' we studied decreased the affinity of ligands for all the three proteins. The substitution of the delta5-3beta-hydroxy grouping for the delta4-3-keto grouping exerted the strongest negative effect in the case of the progesterone receptor and the uterine pentaranophilin, whereas the introduction of the 3',4'-dimethyl grouping strongly inhibited the ligand affinity for the uterine pentaranophilin. The extent and even the direction of the effect of a substituent on the affinity of ligands for the proteins substantially depended on the presence of other substituents in the steroid molecules. We hypothesized that a certain similarity exists between three proteins studied in respect to the structures of their ligand-binding pockets. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

Animals↗

PubMed2004

Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incana R. Br. (Brassicaceae).

For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/anthocyanin biosynthesis pathway belonging to the group of type III polyketide synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in E. Scherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes.

Acyltransferases↗

PubMed2004

Influence of weight and sleep apnea status on immunologic and structural features of the uvula.

We investigated the influence of weight and obstructive sleep apnea status on inflammatory and histologic features of the uvula. Tissue samples resected during uvulopalatopharyngoplasty in 11 snorers without obstructive sleep apnea, 11 subjects with obstructive sleep apnea and of similar body mass index and age, and 8 additional obese subjects with obstructive sleep apnea were examined by immunohistochemistry and histologic staining techniques. The frequency and distribution of immune cells, the amount of collagen, and the integrity of the elastin fiber network were evaluated in proximal and distal uvular sections. T cell (CD4+, CD8+) and macrophage counts were higher in the more obese apneic subjects than in the other two groups. In all patients, T cell counts correlated with body mass index, but there was no relationship with the apnea-hypopnea index. A positive correlation was found between elastin fiber network disorganization score and apnea-hypopnea index. We conclude that (1) the amount of inflammatory markers is linked to obesity rather than to sleep-related breathing disorders, and (2) obstructive sleep apnea is associated with a structural alteration of the extracellular matrix of upper airway tissue.

Biopsy, Needle↗

PubMed2003

Structural features of the acyl chain determine self-phospholipid antigen recognition by a CD1d-restricted invariant NKT (iNKT) cell.

Little is known about the antigen specificity of CD1d-restricted T cells, except that they frequently recognize CD1d-expressing antigen-presenting cells in the absence of exogenous antigen. We previously demonstrated that the 24.8.A iNKT cell hybridoma was broadly reactive with CD1d-transfected cell lines and recognized the polar lipid fraction of a tumor cell extract. In the present study, the antigen recognized by the 24.8.A iNKT cell hybridoma was purified to homogeneity and identified as palmitoyl-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE). The 24.8.A iNKT cell hybridoma recognized synthetic 16:0-18:1[cis] PE, confirming that this phospholipid is antigenic. Recognition correlated with the degree of unsaturation of the acyl chains. Using a panel of synthetic PEs, the 24.8.A iNKT cell hybridoma was shown to be activated by PEs that contained at least one unsaturated acyl chain. The configuration of the double bonds was important, as the 24.8.A iNKT cell hybridoma recognized unsaturated acyl chains in the cis, but not the trans, configuration. PEs with multiple double bonds were recognized better than those with a single double bond, and increasing acyl chain unsaturation correlated with increased binding of PE to CD1d. These data illustrate the potential importance of the acyl chain structure for phospholipid antigen binding to CD1d.

Animals↗

PubMed2004

Structural features of the inactive and active states of the melanin-concentrating hormone receptors: insights from molecular simulations.

Comparative molecular dynamics simulations of both subtypes 1 and 2 of the melanin-concentrating hormone receptor (MCHR1 and MCHR2, respectively) in their free and hormone-bound forms have been carried out. The hormone has been used in its full-length and truncated forms, as well as in 16 mutated forms. Moreover, MCHR1 has been simulated in complex with T-226296, a novel orally active and selective antagonist. The comparative analysis of an extended number of receptor configurations suggests that the differences between inactive (i.e., free and antagonist-bound) and active (i.e., agonist-bound) states of MCHRs involve the receptor portions close to the E/DRY and NPxxY motifs, with prominence to the cytosolic extensions of helices 2, 3, 6, and 7. In fact, the active forms of these receptors share the release of selected intramolecular interactions found in the inactive forms, such as that between R3.50 of the E/DRY motif and D2.40, and that between Y7.53 of the NPxxY motif and F7.60. Another feature of the active forms of both MCHRs is the approach of "helix 8" to the cytosolic extension of helix 3. These features of the active forms are concurrent with the opening of a cleft at the cytosolic end of the helix bundle. For both MCHRs, the agonist-induced chemical information transfer from the extracellular to the cytosolic domains is mediated by a cluster of aromatic amino acids in helix 6, following the ligand interaction with selected amino acids in the extracellular half of the receptor.

Amino Acid Motifs↗

PubMed1999

Structural features and light-dependent changes in the sequence 306-322 extending from helix VII to the palmitoylation sites in rhodopsin: a site-directed spin-labeling study.

Sixteen single-cysteine substitution mutants of rhodopsin were prepared in the sequence 306-321 which begins in transmembrane helix VII and ends at the palmitoylation sites at 322C and 323C. The substituted cysteine residues were modified with a selective reagent to generate a nitroxide side chain, and the electron paramagnetic resonance spectrum of each spin-labeled mutant was analyzed in terms of residue accessibility and mobility. The periodic behavior of these parameters along the sequence indicated that residues 306-314 were in a regular alpha-helical conformation representing the end of helix VII. This helix apparently extends about 1.5 turns above the surface of the membrane, with one face in strong tertiary interaction with the core of the protein. For the segment 315-321, substituted cysteine residues at 317, 318, 320, and 321 had low reactivity with the spin-label reagent. This segment has the most extensive tertiary interactions yet observed in the rhodopsin extra-membrane sequences at the cytoplasmic surface. Previous studies showed the spontaneous formation of a disulfide bond between cysteine residues at 65 and 316. This result indicates that at least some of the tertiary contacts made in the 315-321 segment are with the sequence connecting transmembrane helices I and II. Photoactivation of rhodopsin produces changes in structure detected by spin labels at 306, 313, and 316. The changes at 313 can be accounted for by movements in the adjacent helix VI.

Amino Acid Sequence↗

PubMed1990

Structural features of a water-soluble L-arabino-D-xylan from rye bran.

A water-soluble L-arabino-D-xylan, obtained from the chlorite holocellulose of de-lipidated, de-starched, and de-pectinated rye bran by ammoniacal extraction, was composed of L-Ara and D-Xyl in the molar ratio 7.8:10 and had Mw 36,500 and Mn 26,950. The backbone of the polysaccharide was shown to comprise (1----4)-linked beta-D-Xylp residues, with approximately 41% unsubstituted, approximately 33% 2- or 3-substituted, and approximately 26% disubstituted. Single alpha-L-Araf groups were attached to the xylan core; only a small proportion was 2-, 3-, or 5-linked.

Carbohydrate Sequence↗