bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneous distribution of dopamine D2 receptor mRNA in the rat striatum: A quantitative analysis with in situ hybridization histochemistry
Authors: Szele Francis G. , Artymyshyn Roman , Molinoff Perry B. , Chesselet Marie‐Francoise
Published: May 2005

Dopamine D2 receptor mRNAs have recently been cloned and their gross distribution in the central nervous system described. Quantitative in situ hybridization histochemistry with a cRNA probe complementary to the mRNAs encoding approximately 70% of the third intracellular loop of the rat D2 receptor was performed on sections of rat brain to determine whether differences previously observed in the density of ligand binding sites in subregions of the striatum were related to differences in mRNA levels. Film autoradiographic analysis demonstrated 30% more hybridization signal in the lateral compared to the medial caudate‐putamen, a distribution parallel to that of binding of ligands specific for the D2 receptor. Inspection at the cellular level using emulsion autoradiography also indicated a differential distribution of the D2 receptor mRNA. Fewer positively labelled cells, as well as fewer silver grains per cell, were seen in the medial compared to the lateral half of the striatum. This suggests that the gradient seen in autoradiographic studies of the distribution of D2 receptors is related both to regional differences in D2 mRNA levels and to the density of cells expressing the receptor. In addition, the distribution of cells expressing D2 receptor mRNA in the extrastriosomal matrix was compared to that in striosomes identified by the presence of a high density of 3H‐naloxone binding sites. Labelled cells were mainly found in the matrix (3H‐naloxone binding‐poor) but were also seen in striosomes (3H‐naloxone binding‐rich). The results suggest that differences in levels of D2 binding sites in subregions of the striatum are related to differences in the level of expression of this receptor in intrinsic striatal neurons, suggesting differential regulation of dopamine D2 receptor gene expression in topographically distinct striatal neurons.
Periarterial lymphoid sheath in the rat spleen: A light, transmission, and scanning electron microscopic study
Authors: Sasou Shunichi , Sugai Tamotsu
Published: May 2005

The periarterial lymphoid sheath (PALS) in the rat spleen was studied by light, transmission, and scanning electron microscopy. The PALS was divided into three regions: the central region, peripheral region, and marginal zone bridging channel. In the central region, lymphocytes were easily washed away by perfusion. Large spaces were found between flat reticular cells or in large meshworks of stellate reticular cells; these may be deep lymphatic vessels. True lymphatic vessels were found in the central region near the hilus. In the marginal zone bridging channel, flat reticular cells surrounded the central artery in a circumferential pattern and formed channel‐like spaces between the flat reticular cells. These spaces were connected with the meshwork of the red pulp reticular cells and may be a route for lymphocytes between the deep lymphatic vessels and the red pulp. In the peripheral region of the PALS, it was usually difficult to wash away free cells by perfusion, and free cells were found among the reticular cells. In places in the peripheral region, however, free cells were washed away. It is suggested that the lymph flow may start from the region surrounding the PALS, and that the peripheral region of the PALS may also be another route for lymphocyte migration.
Three‐dimensional structure of cytidine monophosphatase reactive trans‐Golgi elements in spinal ganglion cells of the rat
Authors: Rambourg Alain , Clermont Yves
Published: May 2005

In order to analyse, at the electron microscope level, the three‐dimensional configuration of the trans compartment of the Golgi apparatus rat dorsal root ganglia were treated to demonstrate cytidine monophosphatase (CMPase) activity. The localization of enzymatic activity in the Golgi apparatus varied according to cell types. In type A and C cell, CMPase was exclusively located in the transmost sacculotubular element, whereas in type B cells all the saccules of the stacks forming the Golgi ribbon and the trans‐Golgi networks were impregnated. Numerous dense bodies seen at proximity were also CMPase positive. In 3 μm thick sections of type A cells examined at low magnification, the impregnated element was scattered throughout the cytoplasm and never formed a continuous structure. In type B cells, the strongly reactive trans‐Golgi networks did not follow the entire length of the impregnated Golgi ribbon but were preferentially located in the concavity of its arched portions. At higher magnification and in all cell types some tubular portions of the trans‐Golgi networks took the apperance of spheroidal cage‐like structures, the CMPase positive anastomotic tubules forming the bars of the cage. Anastomotic tubules separated from the trans‐Golgi networks formed fenestrated spheres, while nearby CMPase‐reactive dense bodies exhibited a paler hilus. These observations were taken to indicate that in ganglion cells, some CMPase positive dense bodies, presumably lysosomes, formed by fragmentation of the trans‐Golgi networks.
Influence of retinol on human chondrocytes in agarose culture
Authors: Aulthouse Amy Lynn , Carubelli Cecilia M. , Dow Tod M. , Ziegelmayer Christine , Beck Michael
Published: May 2005

Vitamin A and its congeners, collectively called retinoids, are known to have teratogenic potential and have induced craniofacial and limb malformations in numerous animal species. More importantly, retinoids are recognized as teratogenic to fetuses of pregnant women who have taken such preparations for dermatologic disorders. Information gathered from the study of animal models suggests that retinoids interfere with cartilage differentiation. If chondrogenesis in limb development is disturbed it may contribute to limb reductions and malformations. In vitro studies using various animal systems have shown that cartilage matrix macromolecules are altered to resemble those secreted by mesenchymal cells. The response of human chondrocytes to retinoids in vitro is not known. Culture of human chondrocytes in agarose maintains the cartilage phenotype and therefore serves as a model system to evaluate the influence of retinoids directly on human chondrogenesis. The studies presented in this paper were done to determine if the expression of specific matrix macromolecules of human chondrocytes in agarose culture is altered by retinol treatment. Immunocytochemistry demonstrated enhanced labeling of type I collagen while type II collagen labeling was reduced in cultures treated with retinol. In addition, morphometric analyses indicated a decrease in the size and number of chondrogenic clusters and that individual cells synthesized less alcian blue matrix when compared to parallel control cultures. The size of the proteoglycan monomers, glycosaminoglycan side chains as well as the disaccharide composition were not affected. However, there was a reduction in the quantity of proteoglycan monomers produced.
Localization of the Ca2+‐dependent proteinases and their inhibitor in normal, fasted, and denervated rat skeletal muscle
Authors: Kumamoto Toshihide , Kleese William C. , Cong Jinyang , Goll Darrel E. , Pierce Paul R. , Allen Ronald E.
Published: May 2005

Immunofluorescence and immunogold localization studies show that the two Ca2+‐dependent proteinases (μ‐calpain for the micromolar Ca2+‐requiring proteinase and m‐calpain for the millimolar Ca2+‐requiring proteinase) and their protein inhibitor (calpastatin) are located exclusively intracellularly in normal rat soleus muscle. Quantitative immunogold studies indicate that binding of antibodies to both calpains and to calpastatin is approximately two times greater at the Z‐disk of myofibrils than it is at the I‐band or A‐band regions. Mitochondria and nuclei in muscle cells contain both calpains and calpastatin at concentrations approximately one‐tenth and one‐fifth, respectively, of the concentration at the Z‐disk, as estimated by antibody binding. Very little calpain or calpastatin was seen in the cytoplasmic intermyofibrillar spaces, and most of the calpain and calpastatin in muscle cells is associated with intracellular structures. Immunofluorescence results suggest that concentration of m‐calpain but not μ‐calpain or calpastatin is, in some instances, slightly higher near the intracellular surface of the plasma membrane than elsewhere in the muscle cell. Most m‐calpain, however, is distributed throughout the interior of mature rat skeletal muscle cells. Denervation, or fasting and refeeding increases the concentration of the calpains and calpastatin in the muscle cell but does not change their distribution. Some μ‐and m‐calpain and calpastatin is found extracellularly in denervated soleus muscle or soleus muscle from fasting rats, but the extracellular calparns and calpastatin seem to originate from “leakage” of these proteins out of the cell because serum creatine kinase levels are much higher than normal in denervated or fasting rats.
Heterogeneity of fiber and sarcomere length in the human masseter muscle
Authors: van Eijden Theo M. G. J. , Raadsheer Maarten C.
Published: May 2005

This study deals with regional differences in the architectural design of the human masseter muscle. For a number of defined muscle regions the three‐dimensional corrdinates of origin and insertion points, and the lengths of the muscle fibers and the sarcomeres were determined in the closed jaw position. Measurements were made from cadavers and the data were used as input for a model predicting sarcomere length at other mandibular positions. At a closed jaw average muscle fiber length of the muscle regions ranged between 19.0–30.3 mm. The fibers appeared to be considerably longer (35%) anteriorly than posteriorly in the muscle, and deeply situated fibers were on average 5% shorter than superficially situated ones. Average sarcomere length of the regions ranged between 2.27–2.55 μm, indicating that at a closed jaw position sarcomeres are at suboptimum length and have different positions on the length‐tension curve. In the deep layer of the muscle sarcomeres were significantly shorter (6%) than in the superficial layer. Within the superficial layer sarcomere lengths did not differ significantly, but in the deep layer sarcomeres were shorter (8%) posteriorly than anteriorly in the muscle. The model shows that jaw displacement had a different effect on sarcomere length in the muscle regions. When the jaw was rotated about a transverse axis (open/close rotation) sarcomere excursions were relatively small in the posterior muscle regions and large in the anterior regions. The reverse was true when the jaw was rotated contralaterally about a vertical axis. It is concluded that, due to heterogeneity in fiber and sarcomere lengths, the distribution of maximal isometric tension across the muscle at full effort is not uniform.
Spatial distribution of “tissue‐specific” antigens in the developing human heart and skeletal muscle III. An immunohistochemical analysis of the distribution of the neural tissue antigen G1N2 in the embryonic heart; implications for the development of the atrioventricular conduction system
Authors: Wessels A. , Vermeulen J. L. M. , Verbeek F. J. , Virágh S. Z. , Kálmán F. , Lamers W. H. , Moorman A. F. M.
Published: May 2005

A monoclonal antibody raised against an extract from the Ganglion Nodosum of the chick and designated GIN2 proves to bind specifically to a subpopulation of cardiomyocytes in the embryonic human heart. In the youngest stage examined (Carnegie stage 14, i. e., 4 1/2 weeks of development) these GIN2‐expressing cells are localized in the myocardium that surrounds the foramen between the embryonic left and right ventricle. In the lesser curvature of the cardiac loop this “primary” ring occupies the lower part of the wall of the atrioventricular canal. During subsequent development, GIN2‐expressing cells continue to identify the entrance to the right ventricle, but the shape of the ring changes as a result of the tissue remodelling that underlies cardiac septation. During the initial phases of this process the staining remains recognizable as a continuous band of cells in the myocardium that surrounds the developing right portion of the atrioventricular canal, subendocardially in the developing interventricular septum and around the junction of the embryonic left ventricle with the subaortic portion of the outflow tract. During the later stages of cardiac septation, the latter part of the ring discontinues to express GIN2, while upon the completion of septation, no GIN2‐expressing cardiomyocytes can be detected anymore. The topographic distribution pattern of GIN suggests that the definitive ventricular conduction system derives from a ring of cells that initially surrounds the “primary” interventricular foramen. The results indicate that the atrioventricular bundle and bundle branches develop from GIN2‐expressing myocytes in the interventricular septum, while the “compact” atrioventricular node develops at the junction of the band of GIN2‐positive cells in the right atrioventricular junction (the right atrioventricular ring bundle) and the (“pentrating”) atrioventricular bundle. A “dead‐end tract” represents remnants of conductive tissue in the anterior part of the top of the interventricular septum. The location of the various components of the avian conduction system is topographically homologous with that of the GIN2‐ring in the human embryonic heart, indicating a phylogentically conserved origin of the conduction system in vertebrates.
Anti‐vimentin monocional antibody recognizes a cell with dendritic appearance in the chicken's bursa of fabricius
Authors: Olah Imre , Kendall Cynthia , Glick Bruce
Published: May 2005

The bursa of Fabricius was studied by immunohistochemical method using anti‐vimentin monoclonal antibody (clone 3B4). This monoclonal antibody indentified a vimentin positive cell in the medulla of the bursal follicle. During the first 2 weeks of life the vimentin positive cells located along the corticomedullary border and later became prominent in the medulla with the exception of a narrow zone adjacent to the corticomedullary border. After hatching the accumulation of vimentin‐type intermediate filaments on one side of the nucleus endowed the vimentin positive cells with a polarized appearance. This “cap‐like” vimentin positive area of the cytoplasm determined the position of the major cell process. Within the medulla the Ia positive secretory dendritic cells contained secretory granules in one of the cell processes. The distribution, shape, and polarized appearance of the vimentin positive cells were identical with that of the secretory dendritic cells. Therefore, the anti‐vimentin monoclonal antibody proved to be useful for identification of the bursal secretory dendritic cells. During rapid bursal growth the number of secretory dendritic cells increased, possibly, by proliferation of vimentin negative secretory dendritic cell precursors located along the corticomedullary border.
Glucocorticoid effects on rabbit fetal lung maturation in vivo: An ultrastructural morphometric study
Authors: Snyder Jeanne M. , Rodgers Helen F. , O'Brien Jean A. , Mahli Nancy , Magliato Susan A. , Durham Paul L.
Published: May 2005

Maternal administration of glucocorticoids is known to stimulate fetal lung maturation. In the present study, we used microscopy and stereology to evaluate the morphological effects of maternal glucocorticoid treatment on rabbit fetal lung tissue. Betamethasone was administered to pregnant rabbits on days 25 and 26 of gestation at a dose of 0.2 mg/kg body weight. The animal were sacrificed on day 27 of gestation. Glucocorticoid treatment significantly increased the presumptive airspace in the fetal lung tissue but did not alter the relative proportion of epithelium, connective tissue, or vasculature in the tissue. In addition, glucocorticoid treatment significantly increased the proportion of type II cells in the prealveolar epithelium, increased the rate of phosphatidylcholine synthesis, and increased the content of the major surfactant‐associated protein, SP‐A, in the fetal lung tissue. We could detect no effect of betamethasone on lamellar body crosssectional area, numerical density, or volume density within fetal lung type II cells. Glucocorticoid treatment of the pregnant doe caused a decrease in the volume density of intracellular glycogen and an increase in the volume density of mitochondria in fetal lung type II cells. Betamethasone treatment did not alter the distance between fetal lung epithelial cells and subadjacent connective tissué cells. However, glucocorticoid treatment increased the number of connective tissue foot processes that pierced the epithelial basal lamina. Thus, glucocorticoid treatment of the pregnant doe results in structural changes in the fetal lung tissue, an acceleration of some aspects of type II cell defferentiation, and a concomitant increase in epithelial‐mesenchymal interactions.
Sex determination in humans
Authors: Schafer Alan J. , Goodfellow Peter N.
Published: May 2005

In mammals, the Y chromosome induces testis formation and thus male sexual development; in the absence of a Y chromosome, gonads differentiate into ovaries and female development ensues. Molecular genetic studies have identified the Y‐located testis determining gene SRY as well as autosomal and X‐linked genes necessary for gonadal development. The phenotypes resulting from mutation of these genes, together with their patterns of expression, provide the basis for establishing a hierachy of genes and their interactions in the mammalian sex determination pathway.
Mitochondrial genetics and human disease
Authors: Grossman Lawrence I. , Shoubridge Eric A.
Published: May 2005

Mitochondria contain a molecular genetic system to express the 13 protein components of the electron transport system encoded in the mitochondrial genome (mtDNA). Defects in the function of this system result in some diaseases, many of which are multisystem disorders, prominently involving highly aerobic, postmitotic tissues. These defects can be caused by large‐scale rearrangements of mtDNA, by point mutations, or by nuclear gene mutations resulting in abnormalities in mtDNA. Although any of these mutations would be expected to produce a similar clinical phenotype by compromising oxidative phosphorylation, the surprising and puzzling result is that different clinical phenotypes are generally associated with specific mtDNA mutations. Moreover, the same mutation can produce a distinct clinical phenotype in different individuals or pedigrees. MtDNA rearrangements are also found in aged individuals, but at a subclinical level, suggesting that normal and pathological processes can differ by the effect of genetic or environmental factors on the error rate of mtDNA replication.
Protein conformation in bacterial spinae
Authors: Coombs R. W. , Verpoorte J. A. , Easterbrook K. B.
Published: May 2005

The far uv circular dichroism (CD) and infrared spectra of bacterial spinae are reported. Estimates of the protein secondary structure were obtained by three‐component curve‐fitting methods supplemented by rank and factor analysis of CD data matrices. Native spinae were shown to contain approximately 88% antiparallel β‐sheet, 7% α‐helix, and 5% unordered structure based on estimates using poly(L‐lysine). Basis CD spectra derived from globular proteins were shown to give unreliable estimates.
Sequence dependence of the circular dichroism of synthetic double‐stranded RNAs
Authors: Gray Donald M. , Liu Jung‐Jen , Ratliff Robert L. , Allen Fritz S.
Published: May 2005

We have synthesized and studied the CD spectra of five new double‐stranded RNA polymers: poly[r(A‐G)·r(C‐U)], poly[r(A‐U‐C)·r(G‐A‐U)], poly[r(A‐C‐U)·r(A‐G‐U)], poly[r(A‐A‐C)·r(G‐U‐U)], and poly[r(A‐C‐C)·r(G‐G‐U)]. Together with previously published spectra of seven other RNA sequences, the spectra of these new sequences provide a library sufficient to approximate the spectra of all other RNA sequences by first‐neighbor formulas and, in addition, give four spectra with which we may test the validity of first‐neighbor approximations. (1) We find that the spectra of RNA sequence isomers are very different, but that the spectra essentially do obey first‐neighbor relationships. (2) We have derived tentative first‐neighbor assignments of negative bands at about 295 and 210 nm in the CD spectra. (3) A test of spectral independence shows that among the 12 polymer spectra there are at least seven significant independent spectral shapes, one less than the eight needed to give the most accurate spectral analysis of an unknown RNA sequence for its first‐neighbor frequencies. (4) Spectra are calculated for RNAs of random base composition, approximating natural RNAs having complex sequences. (5) A T‐matrix of spectral components assigned to the first‐neighbor base pairs is derived from 10 of the spectra. This matrix allows an estimation of the CD spectrum of any other known RNA sequence or an analysis of the spectrum of an unknown sequence for its distribution of first‐neighbor base‐pair frequencies. (6) Test analyses of two of the synthetic polymers and of two natural RNAs set a probable limit on the accuracy of first‐neighbor frequency determinations using this T‐matrix. (7) Finally, we summarize in an appendix the melting temperatures for all the RNA and corresponding DNA sequences; it appears that the Tm values of both DNAs and RNAs approximately obey first‐neighbor relationships.
Spectroscopic studies of random chain and α‐helical polypeptides in hexafluoroisopropanol
Authors: Parrish Joseph R. , Blout Elkan R.
Published: May 2005

The infrared, ultraviolet, circular dichroism, and optical rotatory dispersion spectra of five synthetic polypeptides dissolved in hexafluoroisopropanol are reported. This solvent is useful because it dissolves most proteins and non‐ionic polypeptides and also is transparent in spectral regions critical for polypeptide conformational diagnoses. Poly‐γ‐morpholinylethyl‐L‐glutamamide has random chain type spectra in this solvent, whereas the spectra of poly‐γ‐methyl‐L‐glutamate, poly‐L‐methionine, poly‐ε, N‐Carbo‐benzoxy‐L‐lysine, and poly‐L‐homoserine indicate that these four polypeptides are α‐helical. Small but significant variability between the different α‐helical polypeptides is seen in their circular dichroism spectra and optical rotatory dispersions. An argument is presented that these differences may be due to slight geometry differences between different α‐helices.
Light scattering of bovine serum albumin solutions: Extension of the hard particle model to allow for electrostatic repulsion
Authors: Minton Allen P. , Edelhoch H.
Published: May 2005

The light scattering of bovine serum albumin (BSA) has been measured at protein concentration up to 90 g/L and at pH values between 4.4 and 7.6. The dependence of scattering on both protein concentration and pH may be quantitatively accounted for by a simple extension of the hard‐sphere model for protein solutions [Ross, P. D. & Minton, A. P. (1977) J. Mol. Biol. 112, 437–452] allowing for electrostatic repulsions between molecules. According to the extended model, the radius of the effective hard spherical particle representing BSA varies with the net electrical charge of the BSA molecule in a manner which may be calculated from electrostatic theory.
Kinetics of formation of fibrin oligomers. II. Size distributions of ligated oligomers
Authors: Bale Marsha D. , Janmey Paul A. , Ferry John D.
Published: May 2005

Human fibrinogen was treated with thrombin in the presence of fibrinoligase (Factor XIIIa) and calcium ion at pH 8.5, ionic strength 0.45, and the ensuing polymerization was interrupted at various time intervals (t) both before and after the clotting time (tc) by solubilization with a solution of sodium dodecylsulfate and urea. Aliquots of the solubilized protein were subjected to gel electrophoresis on polyacrylamide gels after disulfide reduction by dithiothreitol and on agarose gels without reduction. The degree of γ‐γ ligation was determined from the former and the size distribution of ligated oligomers, for degree of polymerization x from 1 to 10, from the latter. In some experiments, thrombin was inhibited, after partial polymerization, by p‐nitrophenyl‐p′‐guanidinobenzoate. From these, it was concluded that for thrombin concentration ⩽0.013 units/mL and fibrinoligase ⩾30 mg/L, oligomer assembly is rapid compared with peptide A release and ligation is rapid compared with assembly. Under these conditions, the theory of the first paper of this series describes rather well the time dependences of the degree of γ‐γ ligation, the weight fractions of monomer and small oligomers, and the number‐ and weight‐average degrees of polymerization after solubilization of the staggered overlapped assemblies, each of which splits to give two strands of end‐to‐end ligated oligomers. The theory assumes that the second A peptide is released by thrombin more rapidly than the first by a factor q, which, from the experimental data, is determined to be 16. The subsequent assembly into staggered overlapped oligomers follows the statistics of linear polycondesation taking into account the presence of both difunctional and monofunctional combining units. For higher thrombin or lower fibrinoligase concentrations, ligation fails to keep pace with oligomer assembly, and the size distributions after solubilization show a higher proportion of very small and a lower proportion of larger ligated oligomers, owing to separation of the staggered overlapped assemblies into smaller fragments.
Lysinium, argininum, glutamate, and aspartate ions in water solution
Authors: Ranghino Graziella , Clementi Enrico , Romano Silvano
Published: May 2005

SCF‐LCAO‐MO ab initio calculations were carried out for the interaction between a charged amino acid and a water molecule. The results obtained were fitted by an analytical potential function of the atom–atom type, and the corresponding potential surfaces were examined by means of orientationally optimized isoenergy contour maps. Monte Carlo simulations were also carried out on a few selected solute–water clusters at T = 300 K, in order to obtain insight into the solvation structure.
Interaction of poly(X) with poly(U): Alkali metal ion specificity suggests a four‐stranded helix
Authors: Roy Kunal B. , Frazier J. , Miles H. Todd
Published: May 2005

Interaction of poly(X) with poly(U) to form a regular, ordered structure has been investigated by uv, CD, and ir spectroscopy. The XU complex exhibits a marked dependence of Tm on the nature of the alkali metal ion present in solution. Stability of the complex is surprisingly high and increases with ionization of X residues at N3. The ir evidence shows the U residues are present as the diketo form and X residues as the 6‐keto‐2‐enolate anion and indicates that the X and U carbonyl groups are vibrationally coupled. A mixing curve exhibits a single minimum, at 1:1, but we conclude that the actual combining ratio is twice this value and that the formula of the complex is X2U2. We propose a model based on a four‐standard helix with size‐specific alkali metal complexing to X and U carbonyl oxygens in the axial channel of the helix. This model and previously suggested two‐stranded structures are evaluated in terms of the physical and chemical properties of the complex.
Secondary structure of polypeptide hormones of the anterior lobe of the pituitary gland
Authors: Makarov A. A. , Esipova N. G. , Lobachov V. M. , Grishkovsky B. A. , Pankov Yu. A.
Published: May 2005

The specific secondary structure of a number of polypeptide hormones of the pituitary gland anterior lobe and their fragments were studied by CD in the peptide bond absorption region and by ir spectroscopy. The state of objects was examined in solvents of different polarity over a wide temperature range as well as in the solid state at different relative humidities. The predominant conformational state of a number of hormones in aqueous solution is shown to represent a left‐handed helix of the poly(L‐proline) II type. The reversible melting process of the left‐handed helical conformation when heated in an aqueous solution appears to be noncooperative. Lowering the temperature stabilizes the left‐handed structure. The transition mode of the left‐handed form to the α‐, and the β‐forms on changing the solvent conditions was also studied. Contributions of peptide chromophores and of the aromatic amino acid side‐group chromophores with CD bands in the region under study were determined by analysis of CD spectra. The data obtained allow correlating the conformation of separate fragments in the hormone chain with functional activity.
Cyclic peptides revisited
Authors: Madison Vincent
Published: May 2005

A multistep computational scheme was used to deduce possible conformations for a cyclic antagonist analog of somatostatin that has been reported by Coy and coworkers. An algebraic algorithm was used to find dihedral angles that give cyclic structures, the energy was computed for these structures, the lower‐energy structures were classified into conformational families, the energy was minimized for the lowest‐energy member of each family, and finally, the structures from energy minimization were reclassified. Analysis revealed seven distinct conformational families that contain reverse turns. The families differ in the position of the turns in the primary sequence; frame‐shifted turns are observed at each possible position.
Sequence‐Dependence of secondary structure formation: Conformational studies of host–guest peptides in α‐helix and β‐structure supporting media
Authors: Mutter M. , Maser F. , Altmann K.‐H. , Toniolo C. , Bonora G. M.
Published: May 2005

A newly designed host–guest approach is introduced as a experimental tool to explore the relationship between the sequence of peptides and their secondary structure. From the CD spectra of the host–guest peptides studied, a tentative scale for the α‐helix potential in 2,2,2‐trifluorethanol of guest amino acids is delineated. The conformational preferences are also examined in β‐structure supporting media (solid state, CH2Cl2, CH3OH, H2O) using ir‐absorption and CD techniques. Scales for the β‐forming tendency of guest amino acid residues in the different media are delineated. It is shown that the preferred conformation of the host–guest peptides is a function of the medium, the chain length, and the protecting groups. Given the fact that conformational effects are important in peptide synthesis, the tentative scales may serve as a guideline to predict secondary structures of side‐chain‐protected or ‐deprotected peptides in a given solvent, complementing the well‐known empirical conformational prediction parameters.
Molecular recognition. I. Automatic identification of topographic surface features
Authors: Lee Richard H. , Rose George D.
Published: May 2005

The phenomenon of molecular recognition depends strongly on surface complementarity between associating molecular units, analogous to the assembly of a three‐dimensional jigsaw puzzle. The driving force for association is solvent entropy, which increases when protrusions of the irregular surface of one subunit fit snugly into invaginations of the other, squeezing out intervening water. To model this process, an algorithm is presented that automatically finds the prominent topographic surface features of rigid α‐helices in proteins of known structure. When the algorithm was applied to two interacting helices from flavodoxin, only a small number of such features were found, and they accounted for the observed complementarity. Using this algorithm, molecular docking can be reduced to a tractable problem by a docking strategy based on exhaustive trial of combinations of surface features.
β‐Turn structure and intramolecular interaction of tetrapeptides containing Asp and Lys
Authors: Ishii H. , Fukunishi Y. , Inoue Y. , Chûjô R.
Published: May 2005

Nmr and CD studies of terminally protected tetrapeptides were carried out in aqueous and DMSO solutions to investigate the formation and stabilization of the β‐turn structure. Boc‐Gly‐Lys‐Asp‐Gly‐OMe and Boc‐Asp‐Lys‐Asp‐Gly‐OMe appear to have a tendency to adopt a β‐turn structure in aqueous solution from the CD spectra and temperature‐dependence studies of the amide proton chemical shifts. The side‐chain conformation of the Asp residue depends greatly on its ionization state but was not affected by the deprotonation of the neighboring Lys side chain. There is evidence for an intramolecular interaction between the Asp and Lys side chains of Boc‐Gly‐Lys‐Asp‐Gly‐OMe. Such an interaction can contribute to the stabilization of the β‐turn structure.
Thermally induced protein gelation: Gelation and rheological characterization of highly concentrated ovalbumin and soybean protein gels
Authors: Van Kleef F. S. M.
Published: May 2005

In order to optimize the use of proteins as functional ingredients in foods, one needs more insight into the effects of environmental conditions (pH, ionic strength, and temperature) on the functional properties of protein. This paper summarizes the results of an extensive study on heat‐induced gelation of ovalbumin (egg‐white protein) and soybean protein in the concentration range from 10 to 35 g/100 g. It was the aim of the study to relate the rheological properties of thermally induced protein gels to the microstructure of the gel and the physicochemical properties of the constituent protein. The gelling behavior of the protein was quantified with rheological techniques, and the physical properties of the gels were determined, at small and large deformations. From the swelling/dissolving behavior of the gels in various media, the nature of the crosslinks was determined qualitatively. The microstructure of the gels was determined with electron microscopy. Nmr‐spectroscopy was applied in order to elucidate changes in conformation during heating. It was found that the formation of a continuous covalently crosslinked network is not a prerequisite for thermally‐induced protein gelation. The properties of a gel strongly depend on the pH at which the gel is formed. When heat‐set at high pH(pH∼10), a homogeneous, strong, and almost transparent gel is formed, consisting of flexible crosslinked protein gels. Heat‐setting at low pH (pH 5) leads to the formation of a heterogeneous and weak gel, which easily exudes water. This gel consists of crosslinked aggregated protein. The ionic strength of the solvent in which the protein is dissolved and heat‐set has a much lower effect on gel properties.
Unusual conformational transitions of leucine‐containing basic polytripeptides
Authors: Šokrová ŠTěPánka , Bohdanecký Miloslav , Ček Blahoslav Sedlá , Bláha Karel , Šponar Jaroslav
Published: May 2005

The conformational transitions of synthetic basic polytripeptides (Lys‐Leu‐Gly)n, (A2bu‐Leu‐Gly)n, (Lys‐Leu‐Ala)n, and (A2bu‐Leu‐Ala)n induced by high salt concentrations and elevated pH were investigated by CD, ir, and 1H‐nmr spectroscopy, sedimentation analysis, viscometry, and light scattering. Sheet aggregates of chains in a conformation similar to the polyglycine II (polyproline II) helix, bound together by hydrogen bonds, are the most probable form of (Lys‐Leu‐Gly)n and also, partly, of (A2bu‐Leu‐Gly)n in a high‐pH or high‐salt solutions. The conformation (Lys‐Leu‐Ala)n, in a low‐salt concentration, is an α‐helix. Since (A2bu‐Leu‐Ala)n is disordered under similar conditions, it appears that this α‐helix is stabilized by hydrophobic interactions between Lys and Leu side chains. In a high concentration of water structure‐making ions, CD data for (Lys‐Leu‐Ala)n indicate distortion of the α‐helix, with a parallel increase in the average molecular weight corresponding to trimer formation. Hydrodynamic data are consistent with a model of bundles of three closely touching spherocylinders. (A2bu‐Leu‐Ala)n shows a limited tendency to α‐helix formation.
Circular dichroism of double‐stranded RNA in the presence of salt and ethanol
Authors: Steely H. Thomas , Gray Donald M. , Lang Dimitrij , Maestre Marcos F.
Published: May 2005

We have investigated CD spectra of double‐stranded RNA (dsRNA) in a variety of ethanolic and salt solutions. Solution conditions were varied by the addition of salts and ethanol, alone, or in combinations. (1) On addition of either salt or ethanol alone to solutions of dsRNA, we found that the positive CD band at 264 nm increased by up to 7% (in salt) or 12% (in ethanol) and underwent a long‐wavelength shift of 3 nm. Concomitantly, the negative CD band at 296 nm decreased. These CD changes may be indicative of an A‐ to A′‐RNA secondary‐conformation change on dehydration. (2) On addition of both salt and ethanol, in various combinations that caused precipitation of dsRNA, three types of CD spectral changes were found, which we take to indicate at least two different types of intermolecular arrays of RNA molecules. Type I precipitate was flocculent and its CD spectrum had a differential scattering component at long wavelengths. Type II condensate was finely dispersed and was characterized by a doubling of the CD magnitude at 264 nm and simultaneous disappearance of the 210 nm band, to give a CD spectrum close to that previously reported for RNA condensed in the presence of polyethylene glycol and salt [Ψ+ RNA; Evdokimov, Y.M., Pyatigorskaya, T.L., Kadikov, V.A., Polyvtsev, O.F., Doskocil, J., Koudelka, J. & Varshavsky, Y.M., (1976) Nucleic Acids Res. 3, 1533–1547]. Type III precipitate was flocculent and had CD spectra that included features that were present for Types I and II condensates; thus, the RNA in this precipitate may not have been in a unique intermolecular array.
Study of the circular dichroism of bacteriophage ϕ6 and ϕ6 nucleocapsid
Authors: Steely H. Thomas , Gray Donald M. , Lang Dimitrij
Published: May 2005

We have studied the CD of both bacteriophage ϕ6 and ϕ6 nucleocapsid in order to assess the in situ state of the viral, double‐stranded RNA (dsRNA). The results showed that packaged ϕ6 RNA is slightly hyperdichroic (4–5%) at 264 nm relative to dsRNA in solution. Also, the CD of dsRNA within both the virus and the nucleocapsid was unlike CD spectra of any of the three types of dsRNA condensed in vitro, as described in the accompanying paper. Using a curve‐fitting program described elsewhere (Edmondson, S.P. & Gray, D.M. (1983) Nucleic Acids Res. 11, 175–192), we have fit reference CD spectra of ϕ6 RNA and protein to measured CD spectra of ϕ6 intact phage and nucleocapsid. Results of the computer analysis indicated that ϕ6 RNA in situ is spectrally similar to dsRNA that is slightly dehydrated.
23Na‐nmr investigations of counterion exchange reactions of helical DNA
Authors: Braunlin W. H. , Anderson C. F. , Record M. Thomas
Published: May 2005

Changes in Δν½, the nmr linewidth of 23Na, have been determined during titrations of helical DNA with polyamines (divalent putrescine and trivalent spermidine) and with inorganic cations (Mg2+ and Co(NH3)). In each case additions of a multivalent cation (Mz+) to a solution containing NaDNA and NaCl cause decreases in Δν½, which is a population‐weighted average of contributions from nuclei in bound and free environments. Thus, the binding of Mz+ to DNA displaces sodium ions from regions where the quadrupolar relaxation of 23Na is relatively efficient. At a given extent of titration, the binding of a polyamine produces a smaller decrease in Δν½ than does the binding of an inorganic ion of the same valence. The concentration dependence of Δν½ during the course of a titration can be interpreted most simply as a two‐state ion‐exchange reaction by assuming that the binding of Mz does not alter RB, the average relaxation rate of sodium nuclei that remain bound. On the basis of this assumption, the initial linear portions of titration curves can be analyzed to determine upper bounds for r°, the number of sodium ions bound per DNA phosphate in the absence of any competing counterion. Analyzing the titration curves for the four multivalent competitors leads to a range of upper‐bound estimates for r°: 0.5–0.8. The differences in these estimates could indicate that polyamines displace fewer sodium ions from DNA than do their smaller inorganic counterparts. Alternatively, the range in upper‐bound estimates for r° could also reflect specific differences in the effects of the various multivalent cations on RB, if this relaxation rate does change during titration.
1H‐nmr studies of a monointercalating drug into a d[CpGpCpG]2 minihelix
Authors: Delepierre Muriel , Delbarre A. , D'Estaintot B. Langlois , Igolen J. , Roques B. P.
Published: May 2005

The structure of the complex formed between the 7H‐pyridocarbazole monomer [{(2‐piperidyl)‐2,1‐ethane‐yl} {10‐methoxy‐7H‐pyrido[4,3‐c]carbazolium} dimethane sulfonate] and the autocom‐plementary tetranucleotide d(CpGpCpG)2 in aqueous solution is analyzed by 270‐MHz and 400‐MHz 1H‐nmr. The strong upfield shifts observed on most aromatic resonances of both the drug and the nucleotide are interpreted as the result of intercalation of the 7H‐pyridocarbazole monomer in the base‐paired minihelix of d(CpGpCpG). The observation of intermolecular negative nuclear Overhauser effects induced in some drug resonances by irradiation of sugar protons confirms this conclusion. A privileged orientation of the drug in the intercalation site with the quaternizing ethyl piperidine chain protruding in the major groove is proposed.
Interaction of amphipathic polypeptides with phospholipids: Characterization of conformations and the CD of oriented β‐sheets
Authors: Bazzi Mohammed D. , Woody Robert W. , Brack André
Published: May 2005

The effects of interaction with phospholipids on the conformation of the alternating copolymer, poly(Leu‐Lys), and the random copolymer poly(Leu50, Lys50) have been investigated by CD and ir spectroscopy. Poly(Leu‐Lys) undergoes a partial unordered → β‐sheet transition in solution in the presence of lysolecithin. On addition of lysolecithin plus cholate, an unordered → α ‐helix transition is observed. In films deposited from these solutions, poly(Leu‐Lys) adopts the anti‐parallel β‐sheet conformation, as in aqueous solutions at moderate ionic strength. Polarized ir spectra showed that the plane of the β‐sheet in such films deviates from the plane of the film by no more than 14°. The random copolymer, poly(Leu50, Lys50), is α‐helical in the presence of lysolecithin and lysolecithin plus cholate, regardless of whether the sample is a solution or a film. CD measurements on the poly(Leu‐Lys) films provide information about the component of the CD tensor for light propagating normal to the plane of the β‐sheet. These measurements show (1) a negative n → π* CD band (214 nm maximum) with higher intensity than the average CD for isotropic solution; and (2) a positive band in the π → π* region (195 nm maximum), which is weaker than that in the isotropic spectrum.
The effect of methylmercury(II) binding on the conformation of poly(L‐glutamic acid) and poly(L‐lysine): A raman spectroscopic study
Authors: Alex S. , Tajmir‐Riahi H. A. , Savoie R.
Published: May 2005

The binding of the methylmercury cation CH3Hg+ by poly(L‐glutamic acid) (PGA) and by poly(L‐lysine) (PLL) has been investigated by Raman spectroscopy. Coordination on the side‐chain COO− and NH groups of these polypeptides gave characteristic ligand–Hg stretching modes at ca. 505 and 450 cm−1, respectively. Precipitation generally occurred upon formation of the complexes and changes of conformation were common. The solid complex obtained from PGA at pH 4.6 was found to have a mostly disordered conformation, which differed from the respective α‐helical and β‐sheet structures of the dissolved and precipitated uncomplexed polypeptide in the same conditions. An α‐helical structure was generally adopted by the complex formed with PLL, even in pH and temperature conditions where the free polypeptide normally exists in another conformation. The addition of a stronger complexing agent, glutathione, to the PLL/CH3Hg+ complex caused a migration of the bound cations and a restoration of the polypeptide to its original state.
Polynucleotide base‐pair orientation in solution: Linear dichroism and molecular mechanical studies of poly[d(A)]‐poly[d(T)]
Authors: Edmondson Stephen P.
Published: May 2005

Model helices for poly[d(A)]‐poly[d(T)] in solution were constructed based on the base orientations determined by linear dichroism [LD; S. P. Edmondson & W. C. Johnson, Jr. (1985) Biopolymers 24, 824–841] and refined by molecular mechanical energy minimization. The results demonstrate that poly[d(A)]‐poly[d(T)] can form energetically stable conformations with the base orientations measured by LD. Further, only one of the four possible base‐pair orientations that are consistent with the LD results is feasible. Models with negative base tilts had large potential energies, indicating that the LD results do not reflect base motions. The LD models of poly[d(A)]‐poly[d(T)] are stabilized mainly by intrastrand base‐stacking interactions, particularly for the adenine strand.
Conformational heterogeneity of quinoxaline peptides in solution
Authors: Alfredson Thomas V. , Maki August H. , Waring Michael J.
Published: May 2005

The conformational heterogeneity of several quinoxaline antibiotics, a class of naturally occurring quinoxaline peptides with antitumor properties, and their synthetic analogues was investigated in polar and nonpolar solvents by high performance liquid chromatography (HPLC) with uv photodiode array detection, uv‐absorbance, low‐temperature phosphorescence, and nmr techniques. Multiple peak formation and interconversion in the HPLC and 1H‐nmr analysis of triostin A, its under‐N‐methylated synthetic analogues (des‐N‐tetramethyltriostin A [TANDEM] and [N‐MeCys3, N‐MeCys7]‐TANDEM [MCTANDEM]), and echinomycin were examined as a function of temperature, solvent polarity, and residence time in solution prior to analysis. Slow interconversion between HPLC peaks, ascribed to the presence of multiple solution conformers, was exhibited by these peptides although at very different interconversion rates. Among the triostins, the rate of interconversion appeared to vary with the degree of N‐methylation of the residues in the cyclic depsipeptide chain. Interconversion of the n and p conformers of triostin A in chloroform occurred on a chromatographic timescale (a few minutes with kn→p calculated to be 0.02 s−1 at 25°C) while the solution conformers of TANDEM in methanol equilibrated very slowly to one preferred conformer over a period of several weeks at ambient temperature. MCTANDEM, a synthetic analogue of triostin A with an intermediate degree of N‐methylation of the residues in the peptide ring, consisted of an equilibrium mixture of n and p conformers in methanol that interconverted on a chromatographic time scale. Two additional conformers of MCTANDEM developed within a few weeks' residence time in methanol at ambient temperature. Echinomycin was found to exist in methanol as an interconverting mixture of at least four minor conformers in addition to the major isoform (95% by peak area) of the peptide. The solution conformers of the quinoxaline peptides investigated in this report are most likely a consequence of hindered rotation about the N‐methylated peptide bonds in the depsipeptide ring and/or intramolecular hydrogen bonding.