PET-CT在非完全实性肺结节中的应用价值

肺癌在中国恶性肿瘤的发病率位居第一,随着低剂量薄层CT在肺癌筛查中的广泛应用,临床发现更多表现为非完全实性结节的肺腺癌,目前众多研究使CT影像学特征和肺腺癌病理的关系得到更进一步的认知,虽然CT能对部分非完全实性结节做出定性和定位诊断,但仍有部分非完全实性结节诊断困难,PET-CT结合了病灶的代谢信息和精确的定位信息,从而提高对肺部结节诊断的敏感性、特异性、准确性,综合多个文献PET-CT在非完全实性结节中的诊断分期价值较CT无明显提升,却在评估预后和制定合适手术方案上可以起到一定的作用,本文就PET-CT在SSN中的应用价值进行阐述。     

ATP synthesis is driven by an imposed delta pH or delta mu H+ but not by an imposed delta pNa+ or delta mu Na+ in alkalophilic Bacillus firmus OF4 at high pH

Starved whole cells of alkalophilic Bacillus firmus OF4 that are equilibrated at either pH 10.2, 9.5, or 8.5 synthesize ATP in response to a pH gradient that is imposed by rapid dilution of the cyanide-treated cells into buffer at pH 7.5. If a valinomycin-mediated potassium diffusion potential (positive out) is generated simultaneously with the pH gradient, then the rate of ATP synthesis and the level of synthesis achieved is much higher than upon imposition of a pH gradient alone. By contrast, imposition of a large chemical gradient of Na+, either in the presence or absence of a concomitant diffusion potential, fails to result in ATP synthesis. We conclude that this organism does not possess a sodium-motive ATPase that can be made to synthesize detectable levels of ATP by imposition of a suitably large chemical or electrochemical gradient of Na+. On the other hand, a proton-translocating ATPase is in evidence when protons are provided at very high pH, corroborating our earlier work on extremely alkalophilic bacilli. Oxidative phosphorylation must, then, be catalyzed in these organisms by a proton-translocating ATPase even though the putative bulk driving forces for such a catalyst are low under optimal growth conditions. Stable, imposed pH gradients of 1 unit, comparable to the magnitude of the total electrochemical proton gradient of growing cells, result in much lower ATP concentrations than observed in such cells. We hypothesize that ATP synthesis in growing cells utilizes protons that are made available by some localized pathway between proton pumps and the ATP synthase.     

The membrane ATPase of alkalophilic Bacillus firmus RAB is an F1-type ATPase

At the optimal pH for growth (pH 10.5), alkalophilic Bacillus firmus RAB, an obligate aerobe, exhibits normal rates of oxidative phosphorylation despite the low transmembrane proton electrochemical gradient, about -60 mV (delta psi = -180 mV and delta pH = +120 mV). This bioenergetic problem might be resolved by use of an Na+ coupled ATP synthase; otherwise an F1F0-ATPase must be able to utilize low driving forces in this organism. The ATPase activity was extracted from everted membrane vesicles by low ionic strength treatment and purified to homogeneity by hydrophobic interaction chromatography and sucrose density gradient centrifugation. The ATPase preparation had the characteristic F1-ATPase subunit structure, with Mr values of 51,500 (alpha), 48,900 (beta), 34,400 (gamma), 23,300 (delta), and 14,500 (epsilon); the identity of the alpha and beta subunits was confirmed by immunoblotting with anti-beta of Escherichia coli and anti-B. firmus RAB F1. Methanol and octyl glucoside, agents that stimulated the low basal membrane ATPase activity 10- to 12-fold, dramatically elevated the MgATPase activity of the purified F1, more than 150-fold, to 50 mumol min-1 mg protein-1. Anti-F1 inhibited membrane ATPase activity greater than or equal to 80%. The membranes exhibited no Na+-stimulated or vanadate-sensitive ATPase activity when prepared in the absence or presence of Na+ or ATP. These findings, which are consistent with previous studies, establish that in alkalophilic bacteria, ATP hydrolysis, and presumably ATP synthesis is catalyzed by an F1F0-ATPase rather than a Na+ ATPase.     

Buffering capacity of bacilli that grow at different pH ranges.

Cytoplasmic buffering capacities and buffering by whole cells were examined in six bacterial species: Bacillus acidocaldarius, Bacillus stearothermophilus, Escherichia coli, Bacillus subtilis, Bacillus alcalophilus, and Bacillus firmus RAB. Acid-base titrations were conducted on whole cells and cells permeabilized with Triton X-100 or n-butanol. In all of the species examined, the buffering capacity of intact cells was generally a significant proportion of the total buffering capacity, but the magnitude of the buffering capacity varied from species to species. Over the entire range of pH values from 4 to 9.5, B. subtilis exhibited a cytoplasmic buffering capacity that was much higher than that of B. stearothermophilus, B. acidocaldarius, or E. coli. The latter three species had comparable cytoplasmic buffering capacities at pH 4 to 9.5, as long as optimal conditions for cell permeabilization were employed. All of the nonalkalophiles exhibited a decrease in cytoplasmic buffering capacity as the external pH increased from pH 5 to 7. At alkaline pH values, the two thermophiles in the study had particularly low cytoplasmic buffering capacities, and the two alkalophilic bacteria had appreciably higher cytoplasmic buffering capacities than any of the other species studied. Cytoplasmic buffering capacities as high as 1,100 nmol of H+ per pH unit per mg of protein were observed in alkalophilic B. firmus RAB. Since previous studies have shown that immediate cytoplasmic alkalinization occurs upon loss of the active mechanisms for pH homeostasis in the alkalophiles, the very high buffering capacities apparently offer no global protection of internal pH. Perhaps, the high buffering capacities reflect protective mechanisms for specific macromolecules or process rather than part of the mechanisms for bulk pH homeostasis.     

Presence of a nonmetabolizable solute that is translocated with Na+ enhances Na+-dependent pH homeostasis in an alkalophilic Bacillus

The presence of a nonmetabolizable solute whose uptake is coupled to the inward translocation of Na+ has been found to enhance Na+-dependent pH homeostasis and survival of an obligately alkalophilic bacterium. Upon shift of cells of Bacillus firmus RAB from growth medium to buffers at pH 10.5, viability and maintenance of a relatively acidified cytoplasm depended upon the presence of Na+ and was augmented by the inclusion of alpha-aminoisobutyric acid in the buffer. Similarly, when cells were first equilibrated at pH 8.5 and then shifted to buffer at pH 10.5, an extraordinary capacity to maintain a relatively low pHin was exhibited, but only in the presence of Na+. In this protocol, the inclusion of alpha-aminoisobutyric acid actually resulted in an early overshoot of proton influx and also rendered a suboptimal concentration of Na+ efficacious in pH homeostasis. When a protonophoric uncoupler was added to the equilibration and shift buffers, Na+-dependent acidification of the interior was inhibited at early time points. The results support the conclusion drawn from earlier work that a Na+/H+ antiporter plays a critical role in pH homeostasis in the obligately alkalophilic bacilli. Moreover, the current findings indicate that the Na+/solute symporters are a physiologically functional pathway for completing the sodium cycle that controls pHin.     

Purification and characterization of the cytochrome oxidase from alkalophilic Bacillus firmus RAB

A cytochrome oxidase was purified 52-fold from membranes of alkalophilic Bacillus firmus RAB by extraction with Triton X-100, ion-exchange and hydroxyapatite chromatography, and gel filtration. On denaturing gels, the purified enzyme dissociated into two subunits of 56,000 and 40,000 Mr as well as a cytochrome c with an Mr of approximately 14,000. Heme contents calculated for an enzyme with a molecular weight of 110,000 were found to be 2 mol of heme a and 1 mol of heme c per mol of cytochrome oxidase; approximately 2 mol of copper per mol of purified enzyme was also found. Enzyme activity was observed in assays using reduced yeast or horse heart cytochrome c. Activity of the purified enzyme was optimal at pH 6.0 and in the presence of added lipids. Impure, membrane-associated activity exhibited a broader pH range for optimal activity extending to alkaline values.     

Respiratory chain of the alkalophilic bacterium Bacillus firmus RAB and its non-alkalophilic mutant derivative

The membrane-bound respiratory chain components of alkalophilic Bacillus firmus RAB were studied by difference spectroscopy and oxidation-reduction potentiometric titrations. Cytochromes with the following midpoint potentials were identified at pH 9.0: a-type cytochromes, +110 and +210 mV; b-type cytochromes, +20, -120, -280, and -400 mV; and cytochrome c, +60 mV. Only the higher-potential cytochrome a showed an upward shift in midpoint potential when titrated at pH 7.0. Parallel studies of a non-alkalophilic mutant derivative of B. firmus RAB, strain RABN, revealed the presence of only one species each of a-, b-, and c-type cytochromes which exhibited midpoint potentials of +110, -150, and +160 mV, respectively, at pH 7.0. Membranes of both strains were found to contain menaquinone. At pH 9.0, NADH caused the reduction of essentially all of the cytochromes that were seen in fully reduced preparations of wild-type B. firmus RAB membranes. By contrast, at pH 7.0, NADH failed to appreciably reduce the b-type cytochromes. These findings may relate to our recent proposal that an inadequacy in energy transduction (production of a proton motive force) by the alkalophilic respiratory chain at pH 7.0 is what precludes the growth of B. firmus RAB at a neutral pH.     

Mutation of Bacillus firmus OF4 to duramycin resistance results in substantial replacement of membrane lipid phosphatidylethanolamine by its plasmalogen form.

Mutant strains of alkalophilic Bacillus firmus OF4 that were selected for resistance to duramycin had greatly reduced levels of membrane diacylphosphatidylethanolamine, as had been found in studies of such mutants of Bacillus subtilis. In the B. firmus strains, however, substantial levels of plasmenylethanolamine were found. This is an unusual membrane component for an aerobic eubacterium, but the presence of trace amounts even in the wild type was confirmed in experiments with 32Pi-labeled growth medium. The membrane lipid composition of the duramycin-resistant strains had several other changes that also left alkalophilic growth unimpaired.     

Voltage-dependent proton fluxes in liposomes

Liposomes containing buffered KCl were prepared from bacterial lipids, were diluted into K+-free media and were treated with valinomycin to induce the formation of a diffusion potential (delta psi). Upon formation of such a potential, substantial proton influx was observed, as assayed by the quenching of 9-aminoacridine fluorescence. Complete reversal of fluorescence quenching occurred when the potential was collapsed by addition of KCl or when methylamine was added. Studies of proton influx as a function of the theoretical magnitude of the delta psi indicated that the phenomenon occurred only above a delta psi of about -60 mV. Establishment of a Na+ diffusion potential also resulted in proton influx. Treatment of K+-loaded liposomes with N,N'-dicyclohexylcarbodiimide did not reduce the delta psi-dependent proton influx. Moreover, proton influx could be demonstrated upon imposition of a diffusion potential in liposomes prepared from a synthetic lipid. The proton fluxes associated with generation of a diffusion potential in liposomes may complicate studies of reconstituted systems in which proton translocation should occur, and may affect the magnitude of the electrochemical proton gradient that is operant under some conditions.     

Features of apparent nonchemiosmotic energization of oxidative phosphorylation by alkaliphilic Bacillus firmus OF4.

Oxidative phosphorylation by extremely alkaliphilic Bacillus species violates two major predictions of the chemiosmotic hypothesis: the magnitude of the chemiosmotic driving force, the delta p (electrochemical proton gradient), is too low to account for the phosphorylation potentials observed during growth at pH 10.5 without using a much higher H+/ATP stoichiometry than used during growth at pH 7.5, and artificially imposed diffusion potentials fail to energize ATP synthesis above about pH 9.5 (Guffanti, A. A., and Krulwich, T. A. (1989) Annu. Rev. Microbiol. 43, 435-463). To further examine the latter observation, large valinomycin-mediated potassium diffusion potentials were imposed across starved cells of Bacillus firmus OF4 at various pH values from pH 7.5 to 10.5. As the external pH increased above pH 8, there was a sharp decrease in the rate of ATP synthesis in response to an imposed diffusion potential. The rate of ATP synthesis fell to zero by pH 9.2 and 9.4, respectively, in the presence and absence of a small inwardly directed Na+ gradient. Electrogenic Na+/H+ antiport and Na+/alpha-aminoisobutyric acid symport proceeded at substantial rates throughout. When synthesis was energized by an electron donor, cells under comparable conditions synthesized ATP at rapid rates up to pH 10.5. The proton transfers that occur during respiration-dependent oxidative phosphorylation at pH 10.5 may depend upon specific complexes. Cells grown at pH 7.5, which have one-third the levels of the caa3-type terminal oxidase, and slightly lower levels of certain other respiratory chain complexes than pH 10.5-grown cells, support only low rates of ATP synthesis at pH 10.5, although energy-dependent symport and antiport rates are comparable with those in pH 10.5-grown cells. A model is presented for oxidative phosphorylation by the alkaliphilic Bacillus that involves a nonchemiosmotic direct intramembrane transfer of protons from specific respiratory chain complexes to the F0 sector of the ATPase, whereas remaining respiratory chain complexes extrude protons into the bulk to generate the bulk potential required both for ATP synthesis and other bioenergetic work. A pK-regulated gate or a delocalized proton pathway that fails to work above pH 9.5 are suggested as possible features that account for the loss of efficacy of a bulk-imposed diffusion potential in energizing ATP synthesis above pH 9.4.