Ph.D. - Biomedical Sciences (Biophysics)
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ItemInteraction of amino acids and related compounds with neutral polyadenylic acid: a proton magnetic resonance study([Honolulu], 1971)By examining not only proton magnetic resonance (PMR) line separation but also actual chemical shifts of individual aromatic PMR lines of neutral-pH polyriboadenylic acid (poly A), it was established that poly A line separation reflects the degree of base stacking in the single-stranded polynucleotide under a variety of conditions. By monitoring the behavior of poly A in the presence of 19 amino acids and 10 derivatives, it was concluded that a number of aromatic and aliphatic compounds can interact with the polynucleotide via intercalation between adjacent bases, which thereby become destacked. The strength of binding depends primarily on the size, or hydrophobic character, as with the following amino acids: L-Trp>L-Phe>L-His L-Leu >L-Ile>L-Met >L-Vah~L-Pro >L-Ala>Gly but the binding can be aided (as with tryptamine, phenethylamine and histamine) or hindered (as with 3-indolepropionic, 3-phenylpropionic and dihydrourocanic acids) by an electrostatic interaction with the polynucleotide phosphates. It was shown that the PMR melting temperature (T) of 0.04M m - + poly A in 0.2M phosphate buffer, pH =7, was lowered 6±1°C upon addition of 0.15M L-Phe. Other amino acids caused a Tm depression m to an extent reflecting their binding strength. The binding of a number v of compounds was quantitated in terms of association constant K for a 1: 1 interactant: polynucleotide base complex, with the help of a computerized least-squares fit of PMR chemical shifts. Values ranged from K=5-10 M^-1 for L-Trp to KL-Phe> L-His. Negative results were obtained with neutral polycytidylic acid (poly C). The study represents a beginning attempt at understanding the molecular bases which underlie protein--polynucleotide interaction and specificity.
ItemDevelopment of single and double spin-labeling methods and their applications to the study of antibody active sites([Honolulu], 1968)Two specific methods have been developed, utilizing the electron spin resonance (ESR) spin-labeling technique, to probe the dimensional and structural nature of the macromolecular active site and to determine the correlation of active site structure with its biological specificities. The hapten combining sites (HCS) of rabbit anti-dinitrophenyl antibodies have been studied using two classes of twenty-two specifically prepared spin-labeled haptens. The single spin-labeling method employs a hapten labeled with only one spin. The hapten may be either a monovalent or bivalent homologous hapten, or a cross reacting hapten. The line-width and maximum splitting of the anisotropic ESR spectrum of the spin-label is a function of its correlation time Tc. The spectral variation is very sensitive to changes in Tc within the range of 1 to 100 nanoseconds. This range is most suitable for studying dimensional variations of antibody HCS in solution since 100 nanoseconds is approximately the average rotational diffusion time of the antibodies. Pertinent results obtained with this method are: (1) the dominant contribution to the correlation time Tc was found to be associated with freedom of rotation about a single bond for the bound spin label; (2) the average depth of HCS was determined to be 10 Å, and heterogeneity of depth was observed; (3) differences in specificity of antibodies isolated with homologous and cross reacting antigens were found due to structural and dimensional differences at the HCS; (4) homologous haptens form a rigid complex and/or have preferred orientation at HCS, however, cross reacting haptens form a less rigid complex; (5) rotational relaxation time (ρ) for antibody monomer and dimer was determined; (6) a sensitive method has been developed in determining antibody hapten affinity, taking advantage of the complete separation of ESR absorption spectrum of the bound and free spin-label; (7) affinity spin-labels have been demonstrated to be able to distinguish specific and non-specific labeling. Double spin-labeled haptens have two unpaired electrons per molecule. The intensities of the isotropic hyperfine and maximum splitting of the anisotropic ESR spectra are very sensitive to electron-electron exchange and dipolar interactions as well as to the relative orientation to the hyperfine tensors of the two electrons. The anisotropic spectra of the bound labels were only slightly perturbed. This may indicate that the HCS is quite flexible. However, the same spin-labeled hapten containing only one electron was rigidly immobilized at the antibody HCS. From the results of this work, the HCS was found to be dimensionally heterogeneous and probably consists of an inner rigid charge transfer complex region of approximately 5~6 Å with a flexible outer region of 4~5 Å which is partially hydrophobic.
ItemBiophysial studies of nucleosome structure by circular dichroism, thermal denaturation and ESR spin labeling( 1979)Rat liver nuclei were digested very briefly by the Ca^+2, Mg^+2 dependent Endogeneous Endonuclease. The digested chromatin was analyzed using exponential sucrose gradients. By plotting the weight average number of subunits released against digestion time, a slowing in the rate of change of chromatin fragments was found in the region of around six subunits. This suggested that the nucleosomes were possibly folded into a chain of discrete superstructures, with 6 to 8 subunits per superbead, and that the DNA between such superbeads was more susceptible to the nuclease digestion than the linker DNA between the nucleosomes. The conformational state of DNA. in the native chromatin and its subunits was analyzed by thermal denaturation and circular dichroism. Monophasic melting profiles were obtained for both the chromatin and its subunits, suggesting that the electrostatic stabilization of the DNA by the histones (HI, H2A, H2B, H3 and H4) was evenly distributed on the chromatin and its subunits. The chromatin and its subunits showed suppressed DNA ellipticities in their circular dichroism spectra. However, upon assembly of the nucleosomes to form a chromatin fiber, the ellipticity increased until the value of chromatin was achieved. We found that at least 8 nucleosomes were required to give the ellipticity of the chromatin, implying that the nucleosomes were possibly folded in an asymmetric fashion. An imidazole spin label (IMDSL) was used to study the accessibility and conformational state of tyrosines in both the nucleosome core particles and histone core extracted from chicken erythrocytes. About 40% of the tyrosines in the histone core can be labeled under nondenaturing conditions. However, less than 15% of the tyrosines in the nucleosome core particle can be labeled even at 200- to 300-fold molar excess of IMDSL. Conformational changes in the spin labeled histone core and nucleosome core particles due to external perturbations, such as urea, NaCl, pH and temperature, were studied. The nucleosome core was more sensitive than the histone core to urea denaturation. Several conformational transitions in the labeled nucleosome core were observed in the range of 1 mM to 2. 5 M NaCl. A small change was detected at 10 mM NaCl and three major transitions were found between 0.1 M to 0.6 M, 0.7 M to 1.8 M and 2 M to 2.5 M NaCl. The labeled nucleosome core particle appeared to be unaffected by changes of pH in the range of 4.5 < pH < 9.5. A thermal denaturation profile, obtained by the ESR method, showed that gradual conformational changes occurred within the inner histone core before the DNA melted. The mode of reconstitution of nucleosome core particles was studied. A mixture of spin labeled histone cores and core DNA was reconstituted by salt step-gradient dialysis. At each step of dialysis, the labeled proteins were examined by ESR. It was found that the histone core bound progressively to the DNA. in the range of 2 M to 0.3 :M NaCl. Full association between histone core and DNA occurred when the ionic strength was less than 0.3 M. The reconstituted nucleosome complexes, purified by isokinetic sucrose gradients, were found to have identical physical properties as the native particle. The role of tyrosines in the reassociation process for the nucleosome core was also investigated. It was found that spin labeling the surface tyrosines on the histone core did not interfere with proper reassociation of the nucleosome core complex. However, when "buried" tyrosines in the histone core were exposed by urea treatment and then spin labeled with IMDSL, additional histone-DNA complexes were formed with properties different from those of native nucleosome cores. This suggests that some of the "buried" tyrosines are essential for the specific histone-histone interactions leading to the histone core structure.
ItemStudies of the expression of gap junctional protein Connexin43 in dermis and epidermis of human skin : differential modulation by retinoic acid( 1992)Retinoids are effective modulators of proliferation and differentiation of epidermal keratinocytes in vivo and in vitro. Their action in most cases appears due to an alteration in the program of squamous differentiation in normal or diseased epidermal cells. However the mechanism of action of retinoids on skin is not understood at the molecular level. In mouse 10T1/2 cells, the ability of retinoids to suppress neoplastic transformation and enhance growth control is highly correlated with their ability to increase gap junctional intercellular communication and expression of connexin 43. In order to determine if these results have relevance to retinoid action in the human, the interaction of retinoic acid with connexin 43 gene expression in human skin and the potential role of gap junctional communication in the control of differentiation in human skin cells were investigated in the present study. In intact human skin, after two weeks treatment with topical all-trans retinoic acid (RA), levels of Cx43 mRNA and protein increased significantly in epidermis. Immunofluorescence microscopy using an anti Cx43 antibody demonstrated that expression occurred predominantly in the suprabasal layer of epidermis was associated with the thickening of the epidermal layer and the disappearance of the keratinized layer. Furthermore, enhanced Cx43 expression has also been observed in keratinocytes grown on a collagen matrix. Expression was located in suprabasal cells as in intact skin. However, conventional monolayer cultured skin cells are unable to enter a program of normal differentiation and exhibited a differential response to retinoic acid treatment. In keratinocytes increased expression of Cx43 occurred at low (10^-11M ) concentrations whereas inhibition occurred at high (10^-7M ) concentrations, but junctional communication, measured by dye transfer, was not altered over this concentration range. Dermal fibroblasts, in contrast, exhibited a dose-dependent increased expression of Cx43 at concentrations up to 10-7M retinoic acid and' proportionately increased their junctional communication over this dose range. From these observations, it was suggested that regulation of Cx43 gene expression and junctional communication by retinoids in human skin cells is complex. In basal epidermal cells (i.e. those grown on monolayer) Cx43 may not be the major protein involved in intercellular communication. Biochemical changes associated with the differentiation of keratinocytes in the epidermis involve alterations in the pattern of keratin gene expression. Thus keratin gene expression can be used as a marker for determining the process of keratinocyte differentiation. The effect of retinoic acid on keratin 14 expression was studied in 3-dimensional organotypic cultures (raft) where differentiation is allowed. In raft cultures, retinoic acid increased K14 expression. This change of K14 expression by retinoic acid could result from an increase in the number of cells expressing K14. However, it was not shown that the effect of retinoic acid on K14 expression is correlated with Cx43 expression. These data indicate that Cx43 may not be directly involved in the modulation of K14 expression by retinoids. Nevertheless, it may not be excluded that Cx43 plays a role in other aspects of keratinocyte differentiation processes.