Clearance of technetium 99m N-NOET in normal, ischemic-reperfused, and membrane-disrupted myocardium

BACKGROUND: Technetium 99m-labeled bis(N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium(v) (99mTcN-NOET) is a new neutral cardiac perfusion imaging agent that has been shown to have very high uptake and retention in vitro. The purpose of this study was to determine the clearance kinetics of 99mTcN-NOET in control, ischemic-reperfused, and membrane-disrupted myocardium. METHODS AND RESULTS: After a 100 microCi (3.7 x 10(6) Bq) bolus of 99mTcN-NOET was injected, myocardial clearance was monitored for 1 hour by the use of a sodium iodide detector in 30 isolated, Krebs-Henseleit (KH) perfused rat hearts. Seven hearts were used as controls (group 1). In seven ischemic-reperfused hearts, tracer administration and uptake was followed by 30 minutes of no flow and 1 hour of reflow (group 2). In six additional ischemic-reperfused hearts, tracer administration was followed by deprivation of flow for 1 hour followed by 1 hour of reflow (group 3). Six hearts were perfused with a 0.5% Triton X-100 KH perfusate for 1 hour (group 4). Four hearts were perfused with KH for 10 minutes, followed by cyanide for 10 minutes (group 5). This cycle was repeated three times. Activities remaining in each heart at the end of each experiment were quantitated, and activity at peak uptake was calculated. The 99mTcN-NOET myocardial clearance was near linear in the control (0.6 +/- 0.4) and both ischemic-reperfused groups with virtually no fractional clearance (1.2% +/- 0.6% and 2.1% +/- 0.6%, respectively; p = NS). In the Triton X-100 membrane-disrupted hearts, clearance was substantial (94.2% +/- 4.0%; p < 0.0001 compared with the control and ischemic-reperfused groups). Cyanide treatment produced rapid clearance, which was arrested by a return to the standard KH perfusate. Peak uptake as a percentage of injected dose was 74.9% +/- 1.4% for all groups combined. CONCLUSION: Thus 99mTcN-NOET has extremely high myocardial retention after 1 hour in normal myocardium and is not significantly affected by ongoing myocardial ischemia or reperfusion injury in this model. Clearance is increased markedly in extreme conditions of membrane disruption. These data are consistent with the concept that 99mTc-NOET is localized predominantly in or on cell membranes. 99mTcN-NOET is a promising, new myocardial perfusion imaging agent that exhibits a stable myocardial distribution in the setting of acute developing injury.     

Effect of hypotension preceding death on the function of lungs from donors with nonbeating hearts

BACKGROUND: A shortage of suitable brain-dead donors continues to severely limit lung transplantation. Use of donors with nonbeating hearts has been suggested as a solution. Lungs are unique, in that aerobic metabolism can continue in the absence of blood circulation because oxygen is present in airways and alveoli. Animal studies have shown reasonable cadaveric graft function up to several hours after sudden death by drug administration. However, hemodynamic instability before death may worsen lung function through activation and pulmonary sequestration of neutrophils and release of inflammatory mediators. Because many potential cadaveric donors experience hypotension before death, this study was undertaken to assess the effect of hypotensive shock on cadaveric lung viability. METHODS: A rat isolated lung reperfusion model was used to assess pulmonary function over 3 hours of reperfusion or until gross pulmonary edema developed. Twenty-five rats were randomly allocated to the following study groups, which were based on status before lung harvest: (1) control: no interventions; (2) hypotensive: 1 hour of hypotension by exsanguination to a mean blood pressure of 30 to 40 mm Hg; (3) cadaver: death by cervical dislocation followed by 3 hours of in situ lung ischemia; (4) hypotensive + 3 hours cadaver: 1 hour of hemorrhagic shock, followed by death and 3 hours of in situ ischemia; (5) hypotensive + 2 hours cadaver: similar to group 4, except the in situ ischemia was abbreviated to 2 hours. RESULTS: No significant differences were found among group 1, 2, or 3 lungs with regard to wet to dry weight ratios, gas exchange, and pulmonary arterial or airway pressures. However, all group 4 lungs became grossly hemorrhagic and developed severe pulmonary edema within 10 minutes of reperfusion. Group 5 lungs fared only marginally better, with two of five lungs tolerating 3 hours of reperfusion. CONCLUSIONS: A period of hypotension before death severely impairs cadaveric lung viability.     

Analysis of three 2,3-dihydroxybiphenyl 1,2-dioxygenases found in Rhodococcus globerulus P6. Identification of a new family of extradiol dioxygenases

The polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6 contains three bphC genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenases. One of them, bphC1, is clustered with the bphB gene which encodes 2,3-dihydroxy-4-phenylhexa-4,6-diene dehydrogenase and constitutes part of the bph operon specifying the degradation of biphenyl. The nucleotide sequence of bphB and the three bphC genes has been determined. The protein products of the bphBC1 gene cluster were found to exhibit significant homology with the corresponding proteins of analogous degradative pathways in Gram-negative bacteria; the highest homology was in those of the toluene degradation pathway of Pseudomonas putida strain F1. No homology was found between bphC2 and bphC3 and any other sequence in the database. At least two of the three meta cleavage enzymes are inducible by biphenyl. 2,3-Dihydroxybiphenyl 1,2-dioxygenase II, encoded by the bphC2 gene, was purified to apparent homogeneity from a recombinant Escherichia coli strain. The enzyme differed from other extradiol dioxygenases in having a subunit molecular mass of 21 kDa and a hexameric structure. The enzyme contains one tightly bound iron per subunit. These characteristics demonstrate that the 2,3-dihydroxybiphenyl 1,2-dioxygenases encoded by bphC2 and bphC3 belong to a new class of extradiol dioxygenases.     

Triplex formation at physiological pH by oligonucleotides incorporating 5-Me-dC-(N4-spermine)

Oligonucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third strand. While T*A:T triplets are formed at neutral pH, C+*G:C are favoured at acidic pH. Herein it is shown that 18-mer ODN containing spermine conjugated to 5-Me-dC at N4 (1-5), form triplexes with complementary 24-mer duplex 8:9 at neutral pH (7.3, 100 mM NaCl). Under such conditions, control ODN's carrying dC (6) or 5-Me-dC (7) did not show any triple helix formation. Remarkably, the triplexes from spermine-conjugates (1-5) have foremost stability at neutral pH (7.1), unlike the behavior of normal ODN's where optimal stability is at acidic pH (5.5). These results have importance in designing oligonucleotides for antigene applications.     

Cytoplasmic tail deletion of T cell receptor (TCR) beta-chain results in its surface expression as glycosylphosphatidylinositol-anchored polypeptide on mature T cells in the absence of TCR-alpha

Surface expression of the T cell antigen receptor (TCR) in mature T cells requires the association of a variable heterodimer (alpha.beta or gamma.delta) with six invariant CD3 polypeptides (gamma, delta, epsilon-epsilon, zeta-zeta, or zeta-eta). We described here that deletion of the cytoplasmic tail polypeptide sequence (Lys-Lys-Lys-Asn-Ser) of TCR beta-chain (beta CT) results in expression of the truncated beta-chain on the surface of a mature T cell hybridoma line, in the absence of TCR-alpha, as a glycophosphatidylinositol (GPI)-anchored monomeric polypeptide. The GPI-anchored TCR-beta CT is not associated with CD3-epsilon and is incapable of conventional signal transduction. Association with TCR-alpha prevents beta CT from GPI-linkage formation. The alpha beta CT heterodimer binds the CD3 polypeptides, and the resultant TCR alpha beta CT/CD3 complex is capable of signal transduction. Our data show that a signal sequence for GPI-linkage formation is present in TCR-beta, and this alternative membrane anchoring mechanism can be utilized by beta-chain polypeptide lacking the CT sequence. We conclude therefore that in the absence of TCR-alpha expression, the beta-chain CT sequence plays an essential function in hindering GPI-linkage formation, thereby preventing escape of incompletely assembled TCR beta-chain to the cell surface of mature T cells.     

Silica fiber–polybenzoxazine–syntactic foams; Processing and properties

Polybenzoxazine filled with chopped silica fibers and their syntactic foams of varying composition and densities were processed. The composition and density variations were achieved by regulating the relative concentrations of silica fiber and glass microballoons (MB). The variation of tensile, compressive, and flexural properties with change in composition was investigated. For the silica‐fiber filled materials, the property attained a maximum at about 40% volume content of fiber, and thereafter, the properties showed a diminishing trend. The incorporation of microspheres significantly lowered the strength of silica filled materials. However, the decrease in the specific flexural strength was less pronounced and the strength was unaffected beyond a certain microsphere content. During processing and mechanical testing, a large quantity of fibers was fractured, which reduced the strength of silica filled systems. The diminution in material strength on embedding microspheres is attributed to the presence of stress concentrating loci as evidenced from SEM analysis. The various factors leading to the property variation with composition are discussed with microscopic analyses, like clustering of fibers, crack propagation, fiber pull out, and debonding of fibers from resin phase. Dynamic mechanical analysis revealed an improved damping property for the filled materials in contrast to the unfilled polymer. The T_g (deduced from tan δ maximum from DMTA) decreased in silica fiber containing materials and on incorporating the MB, the values reverted back to that of the neat polymer. Both silica and MB conferred better thermal and thermooxidative stabilities to the polybenzoxazine. However, the degradation mechanism is nonoxidative in nature. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008