Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in vitro with rat liver microsomes and nuclei

Loss of tritium from specific positions in [³H,¹⁴C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-³H,¹⁴C]benzo[a]pyrene(B[a]P), [1,3-³H,¹⁴C]B[a]P, [1,3,6-³H,¹⁴C]B[a]P, [6,7-³H,¹⁴C]B[a]P, and [7-³H,¹⁴C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from $\text{[}{}^3\text{H}\text{,}{}^{14}\text{C}\text{]}\text{B}\text{[a]}\text{P}$ was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.     

The structure of two alanine containing ferrichromes: Sequence determination by proton magnetic resonance

Summary Metal coordination confers an extraordinary structural stability to the ferrichromes which, independent of their variable amino acid composition, results in a basically unperturbed conformation for all the homologous peptides in the series. The proton magnetic resonance (pmr) characteristics for Al³⁺ analogues (alumichromes) reflect this conformational isomorphism in usual solvents so that single site substitutions are clearly recognized in the pmr spectra. Thus, the substitution of glycine byl-alanine orl-serine introduce new resonances characteristic of the sidechains and alter the pattern of the amide NH pmr region in that doublets substitute for glycyl triplets at the same site. Since for glycine- andl-serine-containing alumichromes the resonances have already been identified, it is possible to unequivocally establish the primary structure of the twol-alanyl homologues ferrichrome C ( ) and sake colorant A ( ) on the basis of the comparative pmr spectra of their Al³⁺ analogues, namely, alumichrome C and alumisake. The resonance assignment, and hence the site occupancy, is substantiated by the temperature coefficients of the NH chemical shifts, rates of¹H-²H exchange and homonuclear proton spin decoupling experiments centered on the NH spectral region. Occupancy of site 1 by a glycine residue is observed for all known ferrichromes, which serves to conserve a hairpin turn. This method of obtaining sequence information should prove of general use for other systems of homologous polypeptides, provided their conformations are not affected by the residue substitutions.     

The identification of cholesterol and other steroids in Euphorbia pulcherimma

The 4-desmethyl tetracycles of the whole poinsettia plant (Euphorbia pulcherimma) less roots amounted to 0.07% of the wet wt and were shown by ¹H NMR spectroscopy to be steroids and not euphoids. The most abundant component was cholesterol, constituting half the mixture, followed in order of decreasing concentration by 24α-ethylcholesterol (sitosterol), 24α-methylcholesterol (campesterol) and 24β-methylcholesterol (22-dihydrobrassicasterol). The relative amount of cholesterol in this plant is the highest found so far in a tracheophyte. The 4,4-dimethyl compounds (0.1% of wet wt) included lanosterol (5%), 24-dihydrolansterol (5%), β-amyrin (25%), germanicol (50%), an unidentified pentacyclic triterpenoid (8%) and two or more (7%) unidentified components. Both the 4,4-dimethyl- and the 4-desmethylsterols were in the configurational series with a 20α-H-atom. Dihydrolanosterol and lanosterol are the probable intermediates from cycloartenol to cholesterol and 24-alkylcholesterol, respectively. Such a sequence would differ from that operating in most angiosperms, where the alkylation is thought to precede the opening of the 9,19-cyclopropane ring.     

Lectin-Binding Sites Involved in Paramecium primaurelia Mating Pair Formation

ABSTRACT. Mating pair formation in Paramecium primaurelia was shown to be inhibited by incubating mating-competent mating type (mt) I and mt U cells with Limulus polyphemus agglutinin (LPA) or wheat germ agglutinin (WGA). Preincubation of LPA and WGA with their specific binding-monosaccharides, N-acetylneuraminic acid (NeuAc) and N-acetylglucosamine (GlcNAc), respectively, prevented the lectin effect on pair formation. Addition either of NeuAc or GlcNAc resulted in a reversal of cell pairing inhibition by LPA or WGA, respectively. Both NeuAc and GlcNAc monosaccharides inhibited pair formation when their concentration exceeded a threshold value. The pattern of the relative distribution of NeuAc and GlcNAc molecules on the cell surface was analyzed using fluorescence resonance energy transfer techniques combined with imaging systems. Mt I1 cells showed a high lectin-binding site density localized just on the surface region engaged in conjugation. The pattern of mt I cells, consisting of a quite homogeneous lectin-binding site density spread on the cell surface, was also common to nonmating-competent cells and to immature cells. These findings suggest that in P. primaurelia pair formation involves both NeuAc and GlcNAc residues and that the development of mating-competence is related to a modification in NeuAc and GlcNAc relative distribution on the cell surface of mt 11 cells.     

Hen egg white lysozyme fibrillation: a deep‐UV resonance Raman spectroscopic study

Amyloid fibrils are associated with numerous degenerative diseases. The molecular mechanism of the structural transformation of native protein to the highly ordered cross‐β structure, the key feature of amyloid fibrils, is under active investigation. Conventional biophysical methods have limited application in addressing the problem because of the heterogeneous nature of the system. In this study, we demonstrated that deep‐UV resonance Raman (DUVRR) spectroscopy in combination with circular dichroism (CD) and intrinsic tryptophan fluorescence allowed for quantitative characterization of protein structural evolution at all stages of hen egg white lysozyme fibrillation in vitro. DUVRR spectroscopy was found to be complimentary to the far‐UV CD because it is (i) more sensitive to β ‐sheet than to α ‐helix, and (ii) capable of characterizing quantitatively inhomogeneous and highly light‐scattering samples. In addition, phenylalanine, a natural DUVRR spectroscopic biomarker of protein structural rearrangements, exhibited substantial changes in the Raman cross section of the 1000‐cm^–1 band at various stages of fibrillation. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Spin label studies of erythrocytes with abnormal lipid composition: comparison of red cells in a hereditary hemolytic syndrome and lecithin: Cholesterol acyltransferase deficiency

Erythrocytes from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency have been shown to exhibit an increase in membrane fluidity which is surprisingly small in view of the extensive alterations both in membrane lipicl composition (namely, an elevation in cholesterol and phosphatidylcholine contents as well as a decrease in phosphatidylethanolamine) and in the functional properties of these cells. In the hope of deriving some information concerning the interrelationship between the structural and functional abnormalities, we have used the spin probe 5-doxyl stearic acid to investigate the temperature-dependent fluidity properties of red cells from two patients with a hereditary hemolytic syndrome (HHS) whose red cells are also characterized by qualitatively similar alterations in phosphatidylcholine and phosphatidylethanolamine but, unlike those in LCAT deficiency, have relatively normal levels of membrane cholesterol. A small increase in membrane fluidity of HHS erythrocytes equivalent to that previously observed in LCAT deficiency was found, indicating that membrane cholesterol level does not exert an important modulatory influence on membrane fluidity in these cells. It is concluded that while the distinct patterns of structural and functional erythrocyte alterations in these two disorders cannot be explained on the basis of differences in bulk membrane fluidity, the marginally increased fluidity may underlie the abnormalities in osmotic fragility and membrane p-nitrophenylphosphatase activity which are shared in common by both types of modified red cells.     

Investigation of the magnetic susceptibility properties of fresh and fixed mouse heart,liver, skeletal muscle and brain tissue

PurposeSeveral magnetic resonance imaging (MRI) techniques exploit the difference in magnetic susceptibilities between tissues, but systematic measurements of tissue susceptibility are lacking. Furthermore, there is the question as to whether chemical fixation that is used for ex vivo MRI studies, affects the magnetic properties of the tissue. Here, we determined the magnetic susceptibility and water content of fresh and chemically fixed mouse tissue.MethodsMass susceptibility of brain, heart, liver and skeletal muscle samples were determined on a vibrating sample magnetometer at room temperature. Measurements at 50, 125, 200 and 295 K were performed to assess the temperature dependence of susceptibility. Moreover, we measured water content of fresh and fixed samples.ResultsAll samples show mass susceptibilities between −0.068 and −1.929 × 10⁻⁸ m³/kg, compared to −9.338 × 10⁻⁹ m³/kg of double distilled water. Heart tissue has a more diamagnetic susceptibility than the other tissues. Compared to fresh tissue, fixed tissue has a less diamagnetic susceptibility. Fixed tissue was not different in water content to fresh tissue and showed no consistent dependence of susceptibility with temperature, whereas fresh tissue shows a decrease to at least 125 K, indicative of a paramagnetic component.ConclusionsBiological tissues are diamagnetic in comparison to water, where the heart is more diamagnetic than the other tissues, with paramagnetic contributions. Fixation rendered tissue less diamagnetic compared to fresh tissue. Our measurements revealed differences in tissue susceptibility between VSM and QSM, inviting more research to compare susceptibility-based MRI methods with physical measurements of tissue susceptibility.     

NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1)

Human fibroblast growth factor (hFGF-1) is a ∼ 17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu²⁺) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu²⁺ and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu²⁺ appears to stabilize the protein bound to pS vesicles. Cu²⁺ and lipid binding interface mapped using 2D ¹H-¹⁵N heteronuclear single quantum coherence experiments reveal that residues in β-strand I contributes to the unique Cu²⁺ binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and β-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu²⁺, additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu²⁺-induced non-classical secretion of hFGF-1.