Resonance Raman and electron paramagnetic resonance structural investigations of neutrophil cytochrome b558.

The resonance Raman spectra of neutrophil cytochrome b558 obtained upon Soret excitation indicate that the heme is low spin six-coordinate in both ferric and ferrous oxidation states; comparison with the spectra of bis-imidazole hemin suggests imidazole or imidazolate axial ligation. Minor bands attributable to vibrational motions of ring-conjugated vinyl substituents were also observed, consistent with a heme assignment of protoporphyrin IX. The spectra of deoxycholate-solubilized cytochrome b558 were indistinguishable from neutrophil plasma membranes or specific granules, as were spectra from unstimulated and phorbol myristate acetate-stimulated cells, indicating that the hemes are structurally identical in various subcellular environments and cellular physiological states. However, structural complexity was suggested by biphasic ferric-ferrous photoreduction under 413-nm illumination and the absence of an EPR spectrum for the ferric heme under conditions where simple bis-imidazole heme-containing cytochromes are expected to give detectable signals. Midpoint reduction potentials and resonance Raman spectra of the soluble cytochrome b558 from an individual with cytochrome b558 positive (type IA.2) chronic granulomatous disease were nearly identical to normal oxidase, with the exception that the deficient oxidase did not undergo heme photoreduction. Possible structural models are discussed in relation to other physical properties (ligand binding, thermodynamic potentials) exhibited by the cytochrome.     

The complement binding-like domains of the murine homing receptor facilitate lectin activity

The leukocyte homing receptor (HR), the endothelial leukocyte adhesion molecule, and gmp140/platelet activation-dependent granule membrane protein are members of a family of adhesion molecules, termed the lectin cell adhesion molecules (LEC-CAMS) which are unified by a multi-domain structure containing a lectin motif, an epidermal growth factor-like (egf) motif, and variable numbers of a complement binding-like (CB) motif. Previous data have indicated a predominant role for the lectin motif in cell adhesion directed by the LEC-CAMS, although the egf-like domain of the HR may also play a potential role in cell binding. While the role(s) of the CB domains in the LEC-CAMS is currently not understood, they have been hypothesized to act as rigid spacers or stalks for lectin and perhaps, egf domain presentation. In this paper, we analyze the functional characteristics of murine HR-IgG chimeras containing the lectin, lectin plus egf, and lectin plus egf plus CB domains. The Mel 14 mAb, an adhesion blocking antibody which recognizes a conformational determinant in the N-terminus of the HR lectin domain, shows a significantly decreased affinity for a HR construct which lacks the CB motifs, consistent with the possibility that the CB domains are involved with lectin domain structure. In agreement with this conjecture, HR mutants lacking the CB domains show a profound decrease in lectin-specific interaction with the carbohydrate polyphosphomannan ester, suggesting that the changes in Mel 14 affinity for the lectin domain are reflected in lectin functionality. Various assays investigating the interactions between the HR deletion mutants and the peripheral lymph node high endothelium, including cell blocking, immunohistochemical staining, and radioactively labeled ligand binding, all showed that removal of the CB domains results in a lack of HR adhesive function. These results imply that the CB domains of the HR, and, by analogy, the other members of the LEC-CAM family, may play important structural roles involving induction of lectin domain conformation and resultant functionality.     

Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression.

To study the effect of SV40 T-antigen in mammary epithelial cells, a rat beta-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.     

Ligand-dependent synergy of thyroid hormone and retinoid X receptors.

The binding of thyroid hormone receptors to DNA is enhanced by heterodimerization with nuclear proteins. One such heterodimerization partner has recently been characterized as the retinoid X receptor. 9-cis-Retinoic acid has been identified as a natural ligand for retinoid X receptors, suggesting a potential receptor-mediated interaction between thyroid hormone and 9-cis-retinoic acid in the regulation of thyroid hormone-responsive genes. A transient cotransfection assay was used to test for such an interaction. When a complex thyroid hormone response element composed of both direct and inverted repeat hexamers was tested, these two ligands activated gene expression synergistically. In contrast, when the response element consisted only of directly repeated hexamers, unliganded retinoid X receptors enhanced thyroid hormone responsiveness, but 9-cis-retinoic acid induced no additional activation. The results suggest a unique mechanism to achieve differential suggest a unique mechanism to achieve differential thyroid hormone sensitivity of thyroid hormone-responsive genes within a cell. Genes with appropriate response elements will show amplification of the thyroid hormone response by 9-cis-retinoic acid in the presence of retinoid X receptors; other thyroid hormone-responsive genes will be influenced by retinoid X receptors, but not 9-cis-retinoic acid.     

Differential inactivation of Escherichia coli membrane dehydrogenases by a myeloperoxidase-mediated antimicrobial system.

Neutrophil myeloperoxidase, hydrogen peroxide, and chloride constitute a potent antimicrobial system with multiple effects on microbial cytoplasmic membranes. Among these is inhibition of succinate-dependent respiration mediated, principally, through inactivation of succinate dehydrogenase. Succinate-dependent respiration is inhibited at rates that correlate with loss of microbial viability, suggesting that loss of respiration might contribute to the microbicidal event. Because respiration in Escherichia coli can be mediated by dehydrogenases other than succinate dehydrogenase, the effects of the myeloperoxidase system on other membrane dehydrogenases were evaluated by histochemical activity stains of electrophoretically separated membrane proteins. Two bands of succinate dehydrogenase activity proved the most susceptible to inactivation with complete loss of staining activity within 20 min, under the conditions employed. A group with intermediate susceptibility, consisting of lactate, malate, glycerol-3-phosphate, and dihydroorotate dehydrogenases as well as three bands of glucose-6-phosphate dehydrogenase, was almost completely inactivated within 30 min. The relatively resistant group, including the dehydrogenases for glutamate, NADH, and NADPH and the remaining bands of glucose-6-phosphate dehydrogenase, retained substantial amounts of diaphorase activity for up to 60 min of incubation with the myeloperoxidase system. The differential effects of myeloperoxidase on dehydrogenase inactivation could not be correlated with published enzyme contents of flavin or iron-sulfur centers, potential targets of myeloperoxidase-derived oxidants. Despite the relative resistance of NADH dehydrogenase/diaphorase activity to myeloperoxidase-mediated inactivation, electron transport particles prepared from E. coli incubated for 20 min with the myeloperoxidase system lost 55% of their NADH oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spin-trapping of superoxide by 5,5-dimethyl-1-pyrroline N-oxide: application to isolated perfused organs

Of the available techniques used to identify free radicals, spin-trapping offers the unique opportunity to simultaneously measure and distinguish among a variety of important biologically generated free radicals. For superoxide and hydroxyl radical, the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) is most frequently used. However, this nitrone has several drawbacks. For example, its reaction with superoxide is slow, having a second-order rate constant around 10 M-1 s-1. Because of this, high concentrations of DMPO are essential in order to observe the corresponding spin-trapped adduct, 5,5-dimethyl-2-hydroperoxy-1-pyrrolidinyloxy. This may, in some cases, lead to cellular toxicity. In an attempt to circumvent this serious limitation, it has been proposed that an indirect approach be employed to detect and identify free radicals generated as a consequence of ischemia/reperfusion injury. In the direct (most frequently used) approach, the spin trap is first added to an isolated perfused organ under the appropriate experimental conditions. Then, the infusion buffer containing the spin-trap adduct(s) is placed into an quartz flat cell to be inserted into an ESR spectrometer. In the indirect method, the spin trap is added to the perfusate, which had previously exited the organ. Therefore, with this method one can prevent any spin-trap-mediated toxicities to the isolated perfused organ. However, because of the very rapid rate of free radical reactions catalyzed by either superoxide or hydroxyl radical, it is questionable whether ESR spectra recorded using this indirect method result from the actual spin-trapping of free radicals. In this report, we evaluated the indirect spin-trapping technique in light of the kinetic considerations discussed above.     

Postrhinoplasty "red nose": differential diagnosis and treatment by laser

Prior to anticipated nasal surgery, the nasal and facial skin should be examined for any vascular lesions. The skin type should be ascertained. A history of any prior nasal surgery, particularly on the nasal dorsum, should be noted. If rosacea is a clinical possibility, a trial of 1.5 to 2.0 gm q.d. of tetracycline for 6 to 8 weeks is warranted. If, after rhinoplasty, a diffuse "redness" on the nasal dorsum results and one can exclude other diagnoses, then argon laser therapy should be considered. A 3-mm punch biopsy should be obtained to see whether superficial ectatic vessels are present, a finding that would be indicative of a good result from laser therapy.     

Properties and function of the proton-translocating adenosine triphosphatase of Clostridium perfringens.

Growth of Clostridium perfringens was inhibited by compounds which dissipate or prevent the formation of electrochemical proton gradients. Membrane vesicles prepared from this organism exhibited Mg2+-dependent adenosine triphosphatase (ATPase) activity sensitive to N,N'-dicyclohexylcarbodiimide. Mg2+-ATPase activity was optimal of 50 degrees C, but no discrete pH optimum was observed. Adenosine triphosphate-dependent quenching of the fluorescence of the weak base quinacrine by everted membrane vesicles suggested that the Mg2+-ATPase is a proton pump capable of generating an electrochemical proton gradient. Adenosine triphosphate-dependent transport of Ca2+ by everted vesicles was sensitive to uncouplers and inhibitors of the Mg2+-ATPase.