ABSTRACT
An automated method for two-dimensional spatial depiction (mapping) of quantitative physiological tissue characteristics derived from contrast-enhanced MRI was applied to a model of inflammatory disease represented by antigen-induced arthritis of the temporomandibular joint in the rabbit. Specifically, an established two-compartment kinetic model of unidirectional mass transport was implemented on a pixel-by-pixel basis to generate maps of tissue permeability surface area product (PS) and fractional blood volume (BV) based on dynamic MRI intensity data after administration of albumin-(Gd-DTPA)30, a prototype macromolecular contrast medium designed for blood pool enhancement. Maps of PS and BV in a disease model of induced arthritis clearly depicted zones of increased permeability (up to approximately 200 microliters/cc/h-compared to 25 microliters/cc/h in normal tissues).
Subject(s)
Arthritis/pathology , Magnetic Resonance Imaging , Synovial Membrane/blood supply , Temporomandibular Joint/pathology , Animals , Capillary Permeability , Disease Models, Animal , Male , RabbitsABSTRACT
A quick and automated method for quantitative spatial mapping of tissue characteristics derived from contrast enhanced MR imaging by a macromolecular contrast medium (MMCM) was used in normal rats. Specifically, an established two compartment unidirectional flow kinetic model was automatically implemented on a pixel by pixel basis to calculate permeability surface area product (PS) and tissue fractional blood volume (BV) from MRI dynamic intensity data. The utility of PS and BV maps were evaluated in the normal rat abdomen where a range of fractional BV was found: from 100% in the vena cava to 1% in skeletal muscles, with intermediate values for liver and kidney. Tissue permeability depicted on the PS maps was generally low for normal tissues.
Subject(s)
Blood Volume/physiology , Capillary Permeability/physiology , Animals , Contrast Media , Endothelium, Vascular/physiology , Kinetics , Magnetic Resonance Imaging , Models, Biological , Rats , Regional Blood Flow/physiologyABSTRACT
Molecular mechanisms of irreversible thermal inactivation of two bacterial alpha-amylases, from the mesophile Bacillus amyloliquefaciens and from the thermophile Bacillus stearothermophilus, have been elucidated in the pH range of relevance to enzymatic catalysis. At pH 5.0, 6.5, and 8.0, B. amyloliquefaciens alpha-amylase irreversibly inactivates due to a monomolecular conformational process, formation of incorrect (scrambled) structures which subsequently undergo aggregation. At the last pH, this process can be suppressed by the presence of the substrate starch and consequently a covalent process, deamidation of asparagine and/or glutamine residues, becomes the cause of loss of enzymatic activity at 90 degrees C. Monomolecular conformational scrambling is the predominant cause of irreversible inactivation of B. stearothermophilus alpha-amylase at 90 degrees C at pH 5.0, 6.5, and 8.0. At pH 6.5 another contributing inactivation mechanism is the deamidation of amide residues, and at pH 8.0, O2 oxidation of the enzyme's cysteine residue.
Subject(s)
Bacillus/enzymology , Geobacillus stearothermophilus/enzymology , Hot Temperature , alpha-Amylases/metabolism , Acetamides/pharmacology , Amides , Asparagine , Chemical Precipitation , Chromatography, High Pressure Liquid , Cysteine , Enzyme Activation , Glutamine , Half-Life , Hydrogen-Ion Concentration , Isoelectric Focusing , Kinetics , Oxidation-Reduction , Protein Conformation , Sulfhydryl Compounds , ThermodynamicsABSTRACT
Half-lives of Bacillus alpha-amylases at 90 degrees C and pH 6.5 greatly increase in the series from Bacillus amyloliquefaciens to Bacillus stearothermophilus to Bacillus licheniformis, e.g. the difference in thermostability between the first and the third enzymes exceeds 2 orders of magnitude. This stabilization is achieved by lowering the rate constant of monomolecular conformational scrambling, which is the cause of irreversible thermoinactivation of B. amyloliquefaciens and B. stearothermophilus alpha-amylases, so that for B. licheniformis alpha-amylase, another process, deamidation of Asn/Gln residues, emerges as the cause of inactivation. The extra thermostability of the thermophilic enzyme was found to be mainly due to additional salt bridges involving a few specific lysine residues (Lys-385 and Lys-88 and/or Lys-253). These stabilizing electrostatic interactions reduce the extent of unfolding of the enzyme molecule at high temperatures, consequently making it less prone to forming incorrect (scrambled) structures and thus decreasing the overall rate of irreversible thermoinactivation. The implications of these findings for protein engineering are discussed.
