Detecting white matter alterations in multiple sclerosis using advanced diffusion magnetic resonance imaging

Multiple sclerosis is a neurodegenerative and inflammatory disease, a hallmark of which is demyelinating lesions in the white matter. We hypothesized that alterations in white matter microstructures can be non-invasively characterized by advanced diffusion magnetic resonance imaging. Seven diffusion metrics were extracted from hybrid diffusion imaging acquisitions via classic diffusion tensor imaging, neurite orientation dispersion and density imaging, and q-space imaging. We investigated the sensitivity of the diffusion metrics in 36 sets of regions of interest in the brain white matter of six female patients(age 52.8 ± 4.3 years) with multiple sclerosis. Each region of interest set included a conventional T2-defined lesion, a matched perilesion area, and normal-appearing white matter. Six patients with multiple sclerosis(n = 5) or clinically isolated syndrome(n = 1) at a mild to moderate disability level were recruited. The patients exhibited microstructural alterations from normal-appearing white matter transitioning to perilesion areas and lesions, consistent with decreased tissue restriction, decreased axonal density, and increased classic diffusion tensor imaging diffusivity. The findings suggest that diffusion compartment modeling and q-spa ce analysis appeared to be sensitive for detecting subtle microstructural alterations between perilesion areas and normal-appearing white matter.     

Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases

The pH dependence of the pK(a) values of all ionizable groups and of the electrostatic potential at grid points corresponding to catalytically important atoms in the active site of TEM-1 beta-lactamase has been calculated by a mean-field approach for reaction intermediates modeled on the basis of energy minimized x-ray crystallographic coordinates. By estimating electrostatic contributions to the free energy changes accompanying the conversion of the free enzyme into the acylenzyme reaction intermediate, we found that acid-catalyzed protonation of the beta-lactam nitrogen is energetically favored as the initiating event, followed by base-catalyzed nucleophilic attack on the carbonyl carbon of the beta-lactam group. N-protonation is catalyzed through a hydrogen-bonded cluster involving the 2-carboxylate group of the substrate, the side chains of S130 and K234, and a solvent molecule. Nucleophilic attack on the carbonyl carbon is carried out by the side chain of S70 with proton abstraction catalyzed by a water molecule hydrogen-bonded to the side chain of E166. Stabilization of ion pairs in the active site through interactions with distant clusters of charged residues in the enzyme was concluded to be an important driving force of the catalytic mechanism.     

Catalytic and structural role of the metal ion in dUTP pyrophosphatase

The metal ion dependence of the catalytic activity of recombinant Escherichia coli dUTP pyrophosphatase (dUTPase), an essential enzyme preventing incorporation of uracil into DNA, has been investigated by steady-state kinetic, electron paramagnetic resonance, and electron nuclear double resonance methods. Values of k(cat) and k(cat)K(m) were 4.5 +/- 0.1 s(-1) and 0.49 +/- 0.1 x 10(6) M(-1).s(-1) in the absence of divalent metal ions, 14.7 +/- 2.2 s(-1) and 25.1 +/- 7.4 x 10(6) M(-1).s(-1) in the presence of Mg(2+) or Mn(2+), and 24.2 +/- 3.6 s(-1) and 2.4 +/- 0.7 x 10(6) M(-1).s(-1) when supported by VO(2+) or bis(acetylacetonato)oxovanadium(IV). Binding of VO(2+) to the enzyme in the presence of dUDP, a nonhydrolyzable substrate analog, was specific and competitive with Mg(2+). Electron paramagnetic resonance spectra of the ternary enzyme-VO(2+)-chelate-dUDP complex revealed a pattern of (31)P superhyperfine coupling specifying two structurally equivalent phosphate groups equatorially coordinated to the VO(2+) ion. Proton electron nuclear double resonance spectra revealed an equatorial acetylacetonate ligand, indicating that one of the organic ligands had been displaced. By molecular graphics modeling, we show that the diphosphate group of enzyme-bound dUDP is sterically accessible to a hemi-chelate form of VO(2+). We propose a similar location compatible with all kinetic and spectroscopic results to account for the reactivity of VO(2+) and the VO(2+)-chelate in dUTP hydrolysis. In this location the metal ion could promote an ordered conformation of the C-terminal fragment that is obligatory for catalysis but dynamically flexible in the free enzyme.     

Epidermal growth factor receptor signaling is required for microadenoma formation in the mouse azoxymethane model of colonic carcinogenesis

Colonic carcinogenesis involves the progressive dysregulation of homeostatic mechanisms that control growth. The epidermal growth factor (EGF) receptor (EGFR) regulates colonocyte growth and differentiation and is overexpressed in many human colon cancers. A requirement for EGFR in colonic premalignancy, however, has not been shown. In the current study, we used a specific EGFR antagonist, gefitinib, to investigate this role of the receptor in azoxymethane colonic premalignancy. The azoxymethane model shares many clinical, histologic, and molecular features of human colon cancer. Mice received azoxymethane i.p. (5 mg/kg/wk) or saline for 6 weeks. Animals were also gavaged with gefitinib (10 mg/kg body weight) or vehicle (DMSO) thrice weekly for 18 weeks, a dose schedule that inhibited normal receptor activation by exogenous EGF. Compared with control colonocytes [bromodeoxyuridine (BrdUrd), 2.2+/-1.2%], azoxymethane significantly increased proliferation (BrdUrd, 12.6+/-2.8%), whereas gefitinib inhibited this hyperproliferation (BrdUrd, 6.2+/-4.0%; <0.005). Azoxymethane significantly induced pro-transforming growth factor-alpha (6.4+/-1.3-fold) and increased phospho-(active) EGFR (5.9+/-1.1-fold), phospho-(active) ErbB2 (2.3+/-0.2-fold), and phospho-(active) extracellular signal-regulated kinase (3.3+/-0.4-fold) in premalignant colonocytes. Gefitinib inhibited activations of these kinases by >75% (P<0.05). Gefitinib also significantly reduced the number of large aberrant crypt foci and decreased the incidence of colonic microadenomas from 75% to 33% (P<0.05). Gefitinib concomitantly decreased cell cycle-regulating cyclin D1 and prostanoid biosynthetic enzyme cyclooxygenase-2 in microadenomas, suggesting that these regulators are key targets of EGFR in colonic carcinogenesis. These results show for the first time that EGFR signaling is required for early stages of colonic carcinogenesis. Our findings suggest, moreover, that inhibitors of EGFR might be useful in chemopreventive strategies in individuals at increased risk for colonic malignancies.     

Regulation of Rab5 Function during Phagocytosis of Live Pseudomonas aeruginosa in Macrophages

Pseudomonas aeruginosa, a Gram-negative opportunistic human pathogen, is a frequent cause of severe hospital-acquired infections. Effectors produced by the type III secretion system disrupt mammalian cell membrane trafficking and signaling and are integral to the establishment of P. aeruginosa infection. One of these effectors, ExoS, ADP-ribosylates several host cell proteins, including Ras and Rab GTPases. In this study, we demonstrated that Rab5 plays a critical role during early stages of P. aeruginosa invasion of J774-Eclone macrophages. We showed that live, but not heat-inactivated, P. aeruginosa inhibited phagocytosis and that this occurred in conjunction with downregulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and in J744-Eclone cells, ExoS ADP-ribosyltransferase activity caused a more severe inhibition of phagocytosis than ExoS Rho GTPase activity. Furthermore, we found that expression of Rin1, a Rab5 guanine exchange factor, but not Rabex5 and Rap6, partially reversed the inactivation of Rab5 during invasion of live P. aeruginosa. These studies provide evidence that live P. aeruginosa cells are able to influence their rate of phagocytosis in macrophages by directly regulating activation of Rab5.     

Quantitative analysis of water proton spectral lineshape: a novel source of contrast in MRI

Previous work in this laboratory has demonstrated improved anatomic and functional images produced from high spectral and spatial resolution (HiSS) MRI of the water proton signal. The present work tests the hypothesis that different Fourier components of the water resonance represent anatomically and/or physiologically distinct populations of water molecules within each small image voxel. HiSS datasets were acquired from tomatoes and rodent tumors at 4.7 T using echo-planar spectroscopic imaging (spatial and spectral resolutions were 117-150 microm and 1.5-3.1 Hz, respectively). Images of each Fourier component of the water resonance (referred to as Fourier component images, or FCIs) were produced. FCIs at frequencies offset from the peak of the water resonance ('off-peak' FCIs) were compared to images of the Fourier component with largest amplitude, i.e. the water peak-height image. Results demonstrate that off-peak FCIs differ significantly from the water peak-height image and that water resonances are often asymmetric. These results show that water signal at various frequency offsets from the peak of the water resonance come from water molecules in different anatomic/physiologic environments. Off-peak FCIs are a new source of structural and functional information and may have clinical utility.