Biomedical subjects

G J Thomas

Publications and source records attributed to G J Thomas.

At least 73 records · Page 4Linked to original sources

PubMed1997

Solution conformations and interactions of alpha and beta subunits of the Oxytricha nova telomere binding protein: investigation by Raman spectroscopy.

Solution conformations of the alpha and beta subunits of the Oxytricha nova telomere binding protein have been investigated by Raman spectroscopy. Raman spectra have also been obtained for a deletion mutant of the beta subunit, betaC232, which retains the N-terminal domain that is active in ternary complex (alpha:beta:DNA) formation but lacks the C-terminal domain that is active in catalyzing guanine quadruplex formation. The Raman spectra show that alpha, beta, and betaC232 are rich in beta-strand secondary structure ( approximately 40-50%) and turns. The Raman signature of the C-terminal 153 amino acids of beta, generated by subtracting the spectrum of betaC232 (residues 1-232) from that of the full subunit, indicates that the domain active in guanine quadruplex formation contains less beta-strand secondary structure and more irregular structure than the domain active in alpha:beta:DNA formation. Raman markers also provide information about the environments and orientations of several key side chains, including tryptophan residues in N- and C-terminal domains of the beta subunit. Both alpha and beta denature between 30 and 40 degrees C, as evidenced by large changes in Raman bands diagnostic of main chain conformation and side chain environments. The Raman spectrum of an equimolar alpha/beta mixture exhibits no evidence of specific interaction between the subunits; further, the denaturation profile of this mixture is indistinguishable from the sum of denaturation profiles of the constituent subunits, consistent with the absence of appreciable interaction between alpha and beta throughout the range 0-50 degrees C. The present results provide insights into the solution conformations of the Oxytricha telomere binding protein subunits and serve as the basis for future study of subunit interactions with telomeric DNA.

Animals↗

PubMed1997

Structure and interactions of the single-stranded DNA genome of filamentous virus fd: investigation by ultraviolet resonance raman spectroscopy.

The filamentous bacteriophage fd is a member of the Ff class of Inovirus, which includes phages f1 and M13. Ultraviolet resonance Raman (UVRR) spectra of fd have been obtained using excitation wavelengths of 257, 244, 238, and 229 nm. Excitation at 257 nm selectively enhances Raman markers of the packaged single-stranded (ss) DNA genome, while excitation at the shorter wavelengths favors the detection of Raman signals from coat protein aromatics, particularly tryptophan (W26) and tyrosine residues (Y21 and Y24) of the viral coat subunit (pVIII). The principal findings are the following: (1) Distinctive markers of dA, dC, dG, and dT residues of the packaged genome are identified in UVRR spectra of fd excited at 257 and 244 nm, despite the low DNA mass composition (12%) of the virion. (2) Raman bands of the bases of packaged ssDNA show extraordinary resonance Raman hypochromism. Raman intensity losses as large as 80% of the parent DNA nucleotide intensities are observed. This is interpreted as evidence of extensive short-range interactions involving bases of the packaged genome. (3) Conversely, Raman bands of tryptophan and tyrosine residues of the coat protein generally exhibit strong hyperchromism. Typically, Raman markers of the aromatic amino acids are about 3-fold more intense in the UVRR spectrum of fd than in spectra of the free amino acids. The very high Raman cross sections for residues Y21, Y24, and W26 are indicative of unusual hydrophobic environments in the viral assembly. (4) UVRR band shifts that accompany the transfer of fd from H2O to D2O solution indicate that bases of the packaged ssDNA are readily exchanged by the solvent. Similarly, the indole N1H group of W26 is accessible to solvent, as shown by N1H --> N1D exchange in D2O solution. (5) The UVRR markers of the packaged fd genome confirm the conclusion reached previously from off-resonance Raman studies that fd DNA nucleosides favor the C3'-endo/anti conformation, rather than the C2'-endo/anti conformation that is characteristic of the lowest energy structure of DNA. We conclude that nucleoside conformations of the packaged fd genome are influenced by the specific organization of ssDNA and coat protein subunits in the native virion assembly.

DNA, Single-Stranded↗

PubMed1997

Polarized Raman spectroscopy of double-stranded RNA from bacteriophage phi6: local Raman tensors of base and backbone vibrations.

Raman tensors for localized vibrations of base (A, U, G, and C), ribose and phosphate groups of double-stranded RNA have been determined from polarized Raman measurements on oriented fibers of the genomic RNA of bacteriophage phi6. Polarized Raman intensities for which electric vectors of both the incident and scattered light are polarized either perpendicular (I[bb]) or parallel (I[cc]) to the RNA fiber axis have been obtained by Raman microspectroscopy using 514.5-nm excitation. Similarly, the polarized Raman components, I(bc) and I(cb), for which incident and scattered vectors are mutually perpendicular, have been obtained. Spectra collected from fibers maintained at constant relative humidity in both H2O and D2O environments indicate the effects of hydrogen-isotopic shifts on the Raman polarizations and tensors. Novel findings are the following: 1) the intense Raman band at 813 cm(-1), which is assigned to phosphodiester (OPO) symmetrical stretching and represents the key marker of the A conformation of double-stranded RNA, is characterized by a moderately anisotropic Raman tensor; 2) the prominent RNA band at 1101 cm(-1), which is assigned to phosphodioxy (PO2-) symmetrical stretching, also exhibits a moderately anisotropic Raman tensor. Comparison with results obtained previously on A, B, and Z DNA suggests that tensors for localized vibrations of backbone phosphodiester and phosphodioxy groups are sensitive to helix secondary structure and local phosphate group environment; and 3) highly anisotropic Raman tensors have been found for prominent and well-resolved Raman markers of all four bases of the RNA duplex. These enable the use of polarized Raman spectroscopy for the determination of purine and pyrimidine base residue orientations in ribonucleoprotein assemblies. The present determination of Raman tensors for dsRNA is comprehensive and accurate. Unambiguous tensors have been deduced for virtually all local vibrational modes of the 300-1800 cm(-1) spectral interval. The results provide a reliable basis for future evaluations of the effects of base pairing, base stacking, and sequence context on the polarized Raman spectra of nucleic acids.

Bacteriophage phi 6↗

PubMed1997

Mechanisms of virus assembly probed by Raman spectroscopy: the icosahedral bacteriophage P22.

A microdialysis flow cell has been developed for time-resolved Raman spectroscopy of biological macromolecules and their assemblies. The flow cell permits collection of Raman spectra concurrent with the efflux of small solute molecules into a solution of macromolecules and facilitates real-time spectroscopic detection of structural transitions induced by the effluent. Additionally, the flow cell is well suited to the investigation of hydrogen-isotope exchange phenomena that can be exploited as dynamic probes of viral protein folding and solvent accessibility along the assembly pathway. Here, we describe the application of the Raman dynamic probe to the maturation of the icosahedral capsid of bacteriophage P22, a double-stranded DNA virus. The P22 virion is constructed from a capsid precursor (procapsid) consisting of 420 coat subunits (gp5) in an outer shell and a few hundred scaffolding subunits (gp8) within. Capsid maturation involves expulsion of scaffolding subunits coupled with shell expansion at the time of DNA packaging. Raman static and dynamic probes reveal that the scaffolding subunit is highly alpha-helical and highly thermolabile, and lacks a typical hydrophobic core. When bound within the procapsid, the alpha-helical fold of gp8 is thermostabilized; however, this stabilization confers no apparent protection against peptide NH-->ND exchange. A molten globule model is proposed for the native scaffolding subunit that functions in procapsid assembly. Accompanying capsid expansion, a small conformational change (alpha-helix-->beta-strand) is also observed in the coat subunit. Domain movement mediated by hinge bending is proposed as the mechanism of capsid expansion. On the basis of these results, a molecular model is proposed for assembly of the P22 procapsid.

Bacteriophage P22↗

PubMed1997

Integrins and oral cancer.

Integrins are a family of heterodimeric, cation-dependent cell membrane adhesion molecules which mediate cell-cell and cell-matrix interactions. They play a fundamental role in the maintenance of tissue integrity and in the regulation of cell proliferation, growth, differentiation and migration. It is not surprising, therefore, that integrins have been implicated in neoplasia and tumour progression and metastasis. Integrin expression and function are altered in malignant cells, although no specific integrin has been implicated in transformation to the malignant phenotype and changes in integrin expression vary both between and within different tumour types. In oral squamous cell carcinomas there is variable loss or reduced expression of beta 1 integrins and of alpha 6 beta 4, which correlates to loss of basement membrane proteins and is most extensive in poorly differentiated lesions. There are also changes in the repertoire of alpha v integrin expression with de novo expression of alpha v beta 6 which may be important in tumour cell migration. Conversely there is reduced expression of alpha v beta 5. In vitro studies suggest that this integrin may be important in oral neoplasia since alpha v-negative cell lines show a malignant phenotype which can be reversed by transfection of the missing integrin. Because alterations in integrin expression in oral cancers are so variable, it seems unlikely that they will be useful as prognostic markers. However, studies of integrin expression and function are increasing our understanding of cell interactions in oral cancer and may pave the way for novel therapeutic interventions to arrest the progression of individual tumours.

Carcinoma, Squamous Cell↗

PubMed1997

Human neutrophils do not degrade major basement membrane components during chemotactic migration.

At sites of inflammation, circulating neutrophils (PMNs) migrate through microvessel walls into the subendothelial interstitium. While endothelial passage is mediated by adhesion proteins, including those of the integrin, selectin and immunoglobulin superfamily classes, the mechanisms used to cross the subendothelial basement membrane (BM) are unclear. Studies examining tumour cell invasion and lymphocyte extravasation suggest several possible mechanisms, including proteolysis. Different cells, however, may use different mechanisms to effect passage. To examine neutrophil-basement membrane interactions in more detail, human PMNs were embedded within reconstituted BM (Matrigel) and used in migration assays. The integrity of the gel following migration was assessed by assaying for the release of incorporated radiolabelled products and by-immunoblotting for specific matrix molecule epitopes. PMNs migrated through Matrigel in response to the chemotactic peptide FMLP. Degradation products of laminin, heparan sulphate proteoglycan or of gelatin, however, were not detected. In contrast, phorbol ester, which triggers activation without migration, released approximately 40% of incorporated HSPG, 30% of gelatin and 20% of laminin as intact molecules or degraded fragments. Electron microscopy of migrating cells demonstrated pseudopodia associated with channels within the Matrigel. Although the serine proteinase inhibitor DFP, plasma and a specific anti-neutrophil elastase IgG blocked degradation, these agents failed to inhibit migration. Migration was inhibited, however, when the Matrigel concentration was increased to 10 mg/ml. Thus, although PMNs will degrade matrix components they do not do so during migration, and proteolytic remodelling of the BM is not a pre-requisite for neutrophil passage.

Basement Membrane↗

PubMed1996

Orientation of tryptophan-26 in coat protein subunits of the filamentous virus Ff by polarized Raman microspectroscopy.

The Ff filamentous virus, which includes the closely related strains fd, fl and M13, serves as a model for membrane protein assembly and is employed extensively as a cloning vector and vehicle for peptide display. The threadlike virion (approximately 6 x 880 nm) comprises a single-stranded DNA genome sheathed by approximately 2700 copies of a 50-residue alpha-helical subunit, the product of viral gene VIII. The pVIII subunit contains a single tryptophan residue (tryptophan-26) which is essential for assembly. We have employed polarized Raman microspectroscopy to determine the orientation of tryptophan-26 in pVIII subunits of oriented fd fibers. The present application is based upon the transfer of tryptophan Raman tensors from a recent study of N-acetyl-L-tryptophan single crystals [Tsuboi et al. (1996) J. Mol. Struct. 379, 43-50]. The polarized Raman spectra of fd indicate that the plane of the indole ring in each pVIII subunit is close to parallel to the virion axis. In this orientation, the line connecting indole ring atoms N1 and C2 is nearly perpendicular to the virion axis, while the indole pseudo-2-fold axis (a line connecting atom C2 to the midpoint of the C5-C6 bond) is approximately 36 degrees from the virion axis. We have used the present results in combination with preferred tryptophan side-chain torsions [chi 1 (C3-C beta-C alpha-N) and chi 2.1 (C2-C3-C beta-C alpha)] in other proteins and a previously determined experimental value of chi 2.1 in fd [Aubrey. K. L., & Thomas, G. J., Jr. (1991) Biophys, J. 60, 1337-1349] to propose a detailed molecular model for the orientation of the tryptophan-26 side chain in the native virus.

Bacteriophages↗

PubMed1996

Raman spectroscopy of the Ff gene V protein and complexes with poly(dA): nonspecific DNA recognition and binding.

Raman spectra of crystals and solutions of the single-stranded DNA binding protein of bacteriophage Ff (gene V protein, gVp) and of solution complexes of gVp with single-stranded poly-(deoxyadenylic acid) [poly(dA)] reveal the following: (i) The gVp secondary and tertiary structures are similar in solution and in the crystal and are dominated by beta-sheet domains, in agreement with NMR and X-ray findings. (ii) Subunit conformation and side chain environments of gVp are virtually unchanged over a wide range of salt concentration (0 < [NaCl] < 100 mM); however, the solution conformation of poly(dA) exhibits sensitivity to added salt. The perturbed Raman markers indicate subtle changes in helix backbone geometry with accompanying small differences in base stacking as the concentration of NaCl is changed. (iii) In complexes with poly(dA), neither the conformation of gVp nor its side chain environments are altered significantly in comparison to the free protein. This is the case at both high salt (nucleotide-to-subunit binding stoichiometry n = 4) and low salt (n = 3). (iv) The Raman signature of poly(dA) undergoes small perturbations upon gVp binding, indicative of small changes in base stacking and phosphodiester backbone conformation. The present results show that the different stoichiometric binding modes of gVp to poly(dA) are accomplished without significant changes in gVp subunit structure and with only modest changes in the single-stranded poly(dA) ligand. This contrasts sharply with sequence-specific double-stranded DNA binding proteins, such as the phage lambda and D108 repressors, which undergo substantial structural changes upon DNA binding, and which also alter more dramatically the Raman fingerprints of their DNA target sites. Thus, nonspecific and specific nucleic acid recognition modes are distinguishable by Raman spectroscopy. The Raman signature of gVp also allows examination of hydrogen bonding interactions of unique side chains within the hydrophobic core (cysteine 33) and at the binding interface (tyrosine 41). These are discussed in relation to the recently published gVp crystal structure.

Amino Acid Sequence↗

PubMed1996

Subunit orientation in the filamentous virus Ff(fd, f1, M13).

The Ff filamentous viruses (fd, M13, f1) are important models for membrane protein assembly and are used extensively as cloning vectors and vehicles for peptide display. The thread-like virion (approximately 6 nm x 880 nm) comprises a single-stranded DNA genome sheathed by approximately 2700 copies of a 50-residue alpha-helical subunit (pVIII). The average inclination of pVIII subunits from the virion axis has been determined experimentally by polarized Raman microspectroscopy of oriented fd fibers, using the amide I Raman tensors obtained from a peptide single crystal. It is found that the average tilt angle, theta, between the pVIII alpha-helix and the virion axis falls within the range 13< theta <20 degrees.

Bacteriophage M13↗

PubMed1996

Raman signature of the four-stranded intercalated cytosine motif in crystal and solution structures of DNA deoxycytidylates d(CCCT) and d(C8).

The Raman spectral signature of the four-stranded cytosine structure formed by intercalation of two hemiprotonated and parallel-stranded oligodeoxycytidylate duplexes (so-called i motif) has been obtained from the crystal structure of d(CCCT) [Kang, C.H., Berger, I., Lockshin, C., Ratliff, R., Moyzis, R., & Rich, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11636-11640]. Identification of Raman markers diagnostic of the cytosine quadruplex is complemented by results obtained in a pH titration of 2'-deoxycytosine-5'-monophosphate (5'-dCMP) to show that the spectral fingerprint associated with N3 protonation of cytosine is distinct from that of quadruplex formation. The Raman spectrum thus provides a definitive basis for evaluating quantitatively both the extent of cytosine quadruplex formation and the degree of cytosine N3 protonation in DNA. Application to aqueous d(CCCT) and d(C8) demonstrates that the four-stranded intercalated structure is formed by both of these oligodeoxycytidylates in aqueous solution. Whereas both 5'-dCMP and the d(CCCT) quadruplex exhibit a midpoint of titration (apparent pKc) of 4.5 +/- 0.2 at 10 degrees C, cytosine protonation in d(C8) is shifted significantly toward the physiological range, with pKc = 5.8 +/- 0.2. The difference in pKc between the two quadruplexes is equivalent to a free energy difference of 1.7 kcal/mol at 10 degrees C. The present findings extend the library of Raman conformation markers to deoxycytidylate residues in the novel i quadruplex. The significance of these results for probing solution conformations of telomeric DNA sequences is also considered.

Base Sequence↗

PubMed1996

Structural transitions in the scaffolding and coat proteins of P22 virus during assembly and disassembly.

An in vitro system for investigating the assembly of the Salmonella phage P22 has been exploited to elucidate the structural basis of recognition between scaffolding protein (gp8) and coat protein (gp5) subunits of the viral procapsid. Raman spectroscopy and circular dichroism have been employed to examine structural thermostabilities of both gp8 and gp5 in native procapsids, and to characterize structural changes accompanying scaffolding exit, procapsid expansion, and shell disassembly. It is found that the secondary structure of the isolated gp8 subunit is rich in alpha-helix (approximately 40%), is highly thermolabile, and is characterized by noncooperative unfolding (Tm approximately 49 degrees C). Conversely, the procapsid-bound gp8 subunit exhibits stabilization of its alpha-helical secondary structure, characterized by cooperative unfolding. Because cooperative unfolding of gp8 coincides with exit from the procapsid, the present results suggest that unfolding and release are coupled processes. Structural differences between procapsid-free and procapsid-bound gp8 subunits are also apparent in Raman markers which monitor environments of tyrosine and tryptophan side chains. Temperature-resolved Raman spectroscopy of the empty procapsid shell reveals three distinct structural transitions for the gp5 subunits. The first, which occurs between 50 and 65 degrees C, is attributed to shell expansion and results in an increase in beta-strand secondary structure. The two higher temperature transitions, occurring within intervals of 70-80 and 80-95 degrees C, respectively, are attributed to partial unfolding of the shell subunit and subsequent shell disassembly. The same gp5 structure transitions are detected for procapsids which contain scaffolding protein. On the basis of the observed thermodynamic coupling between gp8 unfolding and its release from the procapsid, we propose a model for P22 procapsid assembly. Implications of the model for in vivo assembly of dsDNA viruses are discussed.

Bacteriophage P22↗

PubMed1996

Structure, interactions and dynamics of PRD1 virus I. Coupling of subunit folding and capsid assembly.

Bacteriophage PRD1, which infects Escherichia coli and Salmonella typhimurium, consists of an icosahedral capsid enclosing a membrane-packaged double-stranded DNA genome. The viral shell has been investigated using time and temperature resolved Raman and ultraviolet-resonance Raman spectroscopy to reveal novel features of the capsid structure and its pathway of assembly from P3 subunits. Raman spectra show that the shell is thermostable to 50 degrees C, and disassembles between 50 and 70 C degrees with only a small change in P3 conformation. However, the products of thermal disassembly depend sensitively upon total protein concentration. Characterization by analytical ultracentrifugation indicates that below 8 mg/ml, the purified shell disassembles primarily into P3 trimers; at higher concentrations, larger multimers of P3 are formed. Guanidine hydrochloride (GuHCl) dissociation of the P3 shell yields similar results. Purified P3 trimers, isolated either by heat or GuHCl treatment, exhibit structure sensitivity between 30 and 50 degrees C. Thus, shell disassembly diminishes P3 thermostability. Both the lower temperature transition (30 degrees C to 50 degrees C) of the trimer and the higher temperature transition (50 degrees C to 70 degrees C) of the shell involve a conversion of approximately 5% of the P3 peptide backbone from alpha-helix to beta-strand. Deuterium exchange of the P3 peptide backbone reveals more rapid exchange in the shell than in the trimer, consistent with the observed non-specific polymerization of trimers at high concentration. Conversely, the exchange of indole 1NH groups shows that approximately 65% of tryptophan residues are protected against exchange in the assembled shell. The results suggest a mechanism for shell assembly in which the specific association of trimers into the correct shell architecture involves stabilization of a subunit alpha-helical domain and sequestering of selected side-chains from solvent access. We propose a capsid assembly model which couples P3 shell formation with the final step in folding of the P3 subunit.

Bacteriophages↗

PubMed1996

Structure, interactions and dynamics of PRD1 virus II. Organization of the viral membrane and DNA.

Structure, dynamics and stability of the membrane and double-stranded (ds) DNA genome packaged within the native PRD1 virion have been probed by laser Raman spectroscopy. The Raman signature of PRD1 is complex, but exhibits distinctive marker bands diagnostic of the internal lipid bilayer and dsDNA. The Raman markers demonstrate, respectively, a liquid crystalline lipid phase(L(alpha)) and B DNA conformation throughout the temperature range (5 degrees to 50 degrees) of virion stability. Despite the absence of large scale lipid phase transitions or DNA melting, small temperature-dependent changes in the Raman markers of lipid and DNA are detected, indicating coupling between their structures. Minor deviations of DNA from the canonical B form are imposed by the membrane. The Raman markers indicate further that base stacking and phosphate group interactions of the packaged PRD1 genome differ from those of unpackaged PRD1 DNA. Specific Raman band perturbations are proposed as indicators of DNA-membrane interaction. Hydrogen-deuterium exchange kinetics of packaged and unpackaged PRD1 DNA are indistinguishable, demonstrating that base imino and amino protons are not affected significantly by either the condensation or membrane enclosure associated with DNA packaging. This contrasts with the significant acceleration of base exchanges detected in the packaged DNA of bacteriophage P22, which lacks a viral membrane. The distinctive H-->2H exchange profile of the PRD1 genome, the absence of packaging-induced acceleration of exchange kinetics and the apparent direct interaction between DNA and phospholipids suggest a specific role for the viral membrane in PRD1 assembly. We propose a "membrane-surface-catalyzed" model for dsDNA condensation and organization within the PRD1 virion.

Bacteriophages↗

PubMed1996

Receptor binding sites and uptake activities mediating GABA neurotransmission in chronic alcoholics with Wernicke encephalopathy.

Superior frontal cortex (SFC) and primary motor cortex tissue was obtained at autopsy from thirteen severe chronic alcoholics with neuropathologically confirmed Wernicke Encephalopathy (WE) and 22 controls. Cases with both WE and cirrhosis showed markedly fewer neurones in SFC than did WE cases without cirrhosis. The extent of the apparent neuronal loss corresponded to an increase in post-synaptic GABAA receptor sites, as assessed by the binding of [3H]muscimol to synaptic membranes. Increased [3H]muscimol binding was not accompanied by an increase in 'central-type' benzodiazepine binding sites: as assessed by [3H]flunitrazepam binding, these sites were apparently unaltered, while as assessed by [3H]diazepam binding, they were decreased. The affinities of the two benzodiazepine ligands varied differently with disease. These discrepancies between [3H]flunitrazepam and [3H]diazepam binding could not be accounted for, either by the presence of a second, diazepam-preferring, 'central-type' benzodiazepine binding site, or by loss of 'peripheral-type' sites. The changes in the post-synaptic GABAA-benzodiazepine receptor sites did not reflect any regional, disease-related deficit of afferent GABAergic terminals, as assessed by synaptosomal high-affinity [3H]GABA uptake. On a number of indices, it appears most likely that the data reflect both a loss of receptor sites, and a change in the population of receptor sub-types.

Adult↗

PubMed1996

Vibrational analysis of nucleic acids. IV. Normal modes of the DNA phosphodiester structure modeled by diethyl phosphate.

Raman and ir spectra are reported for diethyl phosphate [(CH3CH2O)2PO2-] and diethyl phosphate isotopomers incorporating carbon-13 at methylene group sites [(CH313CH2O)2PO2-] and deuterium substituents on methyl and methylene carbons [(CH3CD2O)2PO2-, (CD3CH2O)2PO2-, (CD3CD2O)2PO2-]. The vibrational spectra are analyzed to develop a consistent set of assignments for the C-C-O-P(O2-)-O-C-C network, which serves as a model for the nucleic acid phosphodiester backbone. The present study resolves previously conflicting vibrational assignments for the phosphodiester skeleton and provides a firm empirical basis for interpreting conformationally sensitive modes of DNA and RNA. Ab initio vibrational analyses have also been conducted on the above isotopomers of diethyl phosphate in the trans-gauche-gauche-trans conformation, optimized using the 3-21+G* basis set at the restricted Hartree-Fock level. The ab initio calculations are in good agreement with the empirical results, thus strengthening the proposed assignment scheme for Raman and infrared spectra. The present study provides a basis for improvement of empirical force fields utilized in previous normal coordinate analyses of the nucleic acid phosphodiester group.

DNA↗

PubMed1996

The neurochemical pathology of thiamine deficiency: GABAA and glutamateNMDA receptor binding sites in a goat model.

Synaptic plasma membranes were prepared from four cerebrocortical areas from six male Angora goats made chronically thiamine deficient (TD) by the administration of AmproliumTM (600-900 mg/kg daily for 38-44 d). Four male controls were matched for age (27-30 mo). Four different radioligands were used to characterise GABAA and Glu-RNMDA receptor binding sites. There were marked, localised and contrasting changes in motor cortex, with an increase in GABAA and a decrease in Glu-RNMDA binding site densities. Less clearcut changes of a similar nature were seen in visual cortex. There was no variation in the parameters of GABA-activated [3H]diazepam binding between cortical areas in control goats, but there was a reduction in the maximal response to GABA in all areas in TD goats. There were regional variations in glutamate-activated [3H]MK-801 binding in control goat brain, and a non-selectively reduced maximal response in TD. Alterations in these indices of GABA- and glutamate-mediated neurotransmission may underlie the neurological signs of acute thiamine deficiency in these animals.

Alcoholism↗

PubMed1996

Vibrational analysis of nucleic acids. V. Force field and conformation-dependent modes of the phosphodiester backbone modeled by diethyl phosphate.

A generalized valence force field is derived for the diethyl phosphate anion [(CH3CH2O)2PO2-] and its deuterium [(CH3CD2O)2PO2-, (CD3CH2O)2PO2- and (CD3CD2O)2PO2-] and carbon-13 [(CH3 13CH2O)2PO2-] derivatives in the stable trans-gauche-gauche-trans conformation. Normal coordinate analysis of the trans-gauche-gauche-trans conformer, which serves as a structural analog of the nucleic acid phosphodiester group, is based on comprehensive infrared and Raman spectroscopic data and vibrational assignments obtained for the diethyl phosphate anion. The generalized valence force field is in good agreement with the scaled ab initio force field of diethyl phosphate and represents significant improvement over earlier modeling of the phosphodiester moiety with dimethyl phosphate. The conformational dependence of skeletal C-C-O-P(O2-)-O-C-C stretching vibrations is also explored. Starting with the trans-gauche-gauche-trans conformation, the frequency dependence of skeletal stretching modes has been obtained by stepwise rotation of the torsion angles of the P-O and C-O bonds corresponding to nucleic acid torsions alpha (P-O5'), beta (O5'-C5'), epsilon (C3'-O3'), and zeta (O3'-P). Both symmetric and antisymmetric phosphoester stretching modes are highly sensitive to P-O and C-O torsions, whereas symmetric and antisymmetric phosphodioxy (PO2-) stretching modes are less sensitive. The present results provide an improved structural basis for understanding previously developed empirical correlations between vibrational marker bands and nucleic acid backbone conformation.

Models, Chemical↗

PubMed1996

DNA melting investigated by differential scanning calorimetry and Raman spectroscopy.

Thermal denaturation of the B form of double-stranded DNA has been probed by differential scanning calorimetry (DSC) and Raman spectroscopy of 160 base pair (bp) fragments of calf thymus DNA. The DSC results indicate a median melting temperature Tm = 75.5 degrees C with calorimetric enthalpy change delta Hcal = 6.7 kcal/mol (bp), van't Hoff enthalpy change delta HVH = 50.4 kcal/mol (cooperative unit), and calorimetric entropy change delta Scal = 19.3 cal/deg.mol (bp), at the experimental conditions of 55 mg DNA/ml in 5 mM sodium cacodylate at pH 6.4. The average cooperative melting unit (nmelt) comprises 7.5 bp. The Raman signature of 160 bp DNA is highly sensitive to temperature. Analyses of several conformation-sensitive Raman bands indicate the following ranges for thermodynamic parameters of melting: 43 < delta HVH < 61 kcal/mol (cooperative unit), 75 < Tm < 80 degrees C and 6 < (nmelt) < 9 bp, consistent with the DSC results. The changes observed in specific Raman band frequencies and intensities as a function of temperature reveal that thermal denaturation is accompanied by disruption of Watson-Crick base pairs, unstacking of the bases and disordering of the B form backbone. These three types of structural change are highly correlated throughout the investigated temperature range of 20 to 93 degrees C. Raman bands diagnostic of purine and pyrimidine unstacking, conformational rearrangements in the deoxyribose-phosphate moieties, and changes in environment of phosphate groups have been identified. Among these, bands at 834 cm-1 (due to a localized vibration of the phosphodiester group), 1240 cm-1 (thymine ring) and 1668 cm-1 (carbonyl groups of dT, dG and dC), are shown by comparison with DSC results to be the most reliable quantitative indicators of DNA melting. Conversely, the intensities of Raman marker bands at 786 cm-1 (cytosine ring), 1014 cm-1 (deoxyribose ring) and 1092 cm-1 (phosphate group) are largely invariant to melting and are proposed as appropriate standards for intensity normalizations.

Animals↗