Mitochondrial depolarization promotes apoptotic and necrotic cell death and possibly other cellular events. Polarized mitochondria take up cationic tetramethylrhodamine methylester (TMRM), which is released after depolarization. Thus, TMRM does not label depolarized mitochondria. To identify both polarized and depolarized mitochondria in living cells, cultured rat hepatocytes, and sinusoidal endothelial cells were co-loaded with green-fluorescing MitoTracker Green FM (MTG) and red-fluorescing TMRM for imaging by laser scanning confocal microscopy. Like TMRM, MTG is a cationic fluorophore that accumulates electrophoretically into polarized mitochondria. Unlike TMRM, MTG binds covalently to intramitochondrial protein thiols and remains bound after depolarization. In cells labeled only with MTG, excitation with blue (488 nm) light yielded green but almost no red fluorescence. After subsequent loading with TMRM, green MTG fluorescence became quenched. Instead, blue excitation yielded red fluorescence. Mitochondrial de-energization restored green fluorescence and abolished red fluorescence. Conversely, when MTG was added to TMRM-labeled cells, red fluorescence excited by blue light was enhanced, an effect again reversed by de-energization. These observations of reversible quenching of donor fluorescence and augmentation of acceptor fluorescence signify fluorescence resonance energy transfer (FRET). In undisturbed hepatocytes, spontaneous depolarization of a subfraction of mitochondria was an ongoing phenomenon. In conclusion, confocal FRET discriminates individual depolarized mitochondria against a background of hundreds of polarized mitochondria.

The recombinant neuraminidases (rNAs) from A/New Caledonia/20/99 (NC) (H1N1), A/PR/8/34 (PR8) (H1N1), A/Panama/2007/99 (Panama) (H3N2) and B/Shangdong/7/97 (B/S) viruses were produced by the CHO-K1 cells, prepared from clones which were selected based on the high expression of NA on the cell surface. Each rNA in the cell lysate was purified by an immuno-affinity column. The purified rNAs were obtained at the levels of approximately 0.6–1.8 mg/l medium. Properties of the recombinant N1-NA from NC virus was compared with other rNAs from PR8, Panama and B/S viruses, because N1-NA remained to be characterized. The results demonstrated that the immunogenicity of N1-rNA molecules, such as NC-NA and PR8-NA, was conserved partially even under the absence of the NA enzymatic activity, although the N1-rNA molecules were more labile than Panama-N2-rNA and B/S-rNA. They also suggested that N1-rNAs could be used as a useful protective antigen, capable of producing cross-protection among N1 influenza viruses when administered intranasally.

The clinical pharmacology of morphine is complicated by its active metabolite, morphine-6-β-glucuronide (M6G). M6G is a potent μ-opioid agonist that has been recognized to play an important role in the clinical effects of morphine. However, M6G probably crosses the blood brain barrier with difficulties, and is of importance for the effects of morphine only during long-term morphine administration because only then M6G may reach high enough CNS concentrations. Since M6G is eliminated from the body via the kidney, it may cause severe opioid side effects with insidious onset and long persistence when renal function is impaired. This time dependent participation of M6G at the clinical effects of morphine makes it difficult to predict the effect of morphine in an individual patient. The problem may be solved using a PK/PD modeling approach to the clinical pharmacology of morphine that takes the delayed action of M6G into consideration.

Using confocal microscopy, we investigated mechanisms underlying loss of plasma membrane integrity during necrotic death of cultured hepatic sinusoidal endothelial cells exposed to 2.5 mM potassium cyanide (chemical hypoxia). After 2–3 h, the anionic fluorophore calcein abruptly began to enter the cytosol, and nuclei labeled with cationic propidium after another 2–5 min. As calcein permeated, growth of blebs on the plasma membrane accelerated. Lucifer yellow, another anionic fluorophore, entered identically to calcein, whereas high molecular weight dextrans (40–2000 kDa) entered like propidium. Glycine slowed, but did not prevent calcein entry, whereas permeation of propidium and high molecular weight dextrans was blocked completely by glycine. These findings suggest that opening of a glycine-sensitive organic anion channel, or death channel, precipitates a metastable state characterized by rapid cell swelling and bleb growth. This metastable state culminates in non-specific breakdown of the plasma membrane permeability barrier and irreversible cell death. Cell Death and Differentiation (2001) 8, 850–858

In this study, we investigate the application of the fuzzy clustering to the anatomical localization and quantitation of brain lesions in Positron Emission Tomography (PET) images. The method is based on the Fuzzy C-Means (FCM) algorithm. The algorithm segments the PET image data points into a given number of clusters. Each cluster is an homogeneous region of the brain (e.g. tumor). A feature vector is assigned to a cluster which has the highest membership degree. Having the label affected by the FCM algorthm to a cluster, one may easily compute the corresponding spatial localization, area and perimeter. Studies concerning the evolution of a tumor after different treatments in two patients are presented.

Region growing is a very useful technique for image segmentation. Its efficiency mainly depends on its aggregation criterion. In the present paper, a new algorithm is proposed with a homogeneity criterion based on an adequate tuning between spatial neighbourhood and histogram neighbourhood. It differs from other techniques by reconsidering the pixel (or voxel) assignments on each step by a process which minimizes variance through special dilations. Thus, the region created by an initial seed can be non-connected and possibly does not contain this seed. Examples are given in dental surgery for 2D X-Ray images (and their associated 3D block) and for 3D images acquired by the Morphometre, the new 3D scanner constructed by GEMSE (General Electric Medical Systems).

To determine whether cure of Helicobacter pylori infection influences the expression of COX-2 and nitrotyrosine in the distal stomach of humans, biopsy specimens were examined immunohistochemically. H. pylori infection was determined using a rapid urease test, culture and histology. Positive staining of COX-2/nitrotyrosine in the epithelium was expressed as the percentage of stained cells to the total epithelial cells. There was a significant increase in COX-2/nitrotyrosine staining in H. pylori -positive subjects compared with H. pylori -negative subjects. Cure of the infection resulted in a significant decrease in both COX-2/nitrotyrosine staining in all patients (52.1+/-12.1% vs 15. 4+/-7.2%, P<0.001; and 57.3+/-13.6% vs 36.1+/-18.0%, P<0.01, respectively). However, immunoreactivity of COX-2/nitrotyrosine was observed in all cases with intestinal metaplasia even after the cure of H. pylori infection.Thus, cure of H. pylori infection may decrease the risk of gastric carcinogenesis due to COX-2 and NO-related compounds in gastric mucosa but not in those patients with intestinal metaplasia.

ABSTRACTTuberculosis osteitis is mainly observed as a late complication of the pulmonary infection. We describe a 9‐mo‐old Japanese infant who became infected with Mycobacterium tuberculosis even after receiving Bacillus Calmette‐Guérin (BCG) vaccination at 3 mo of age. This is the first report that the sternum is the localizatio n for tuberculou s osteitis in infants. He developed a localized tumor in the sternum without any respiratory symptom. Mycobacterium tuberculosis complex was detected by means of nucleic acid amplification test. Gastric aspirates also yielded Mycobacterium tuberculosis. The source of the infection was unclear. Conclusion: Tuberculous osteitis should be excluded in infants with undiagnosed bone lesions, even if they have been vaccinated with BCG. ±BCG vaccination, infant, Mycobacterium tuberculosis, sternum, tuberculous osteitis

The liver sinusoidal endothelial cells (SECs) possess unique hyaluronan receptors that recognize and internalize hyaluronic acid(HA). This characteristic was used in the development of a system for targeting foreign DNA to the SEC.A gene carrier system was prepared by coupling HA (number-average molecular weight: 1.5 x 10(4)) to poly-l-lysine (PLL, number-average molecular weight: 4.6 x 10(4)) in a 1:1 weight ratio by reductive amination reaction. The resulting copolymer (hyaluronate-grafted poly-(L-Lysine): PLL-g-HA) was isolated and lyophilized. The PI,L-g-HA thus formed was mixed with various amounts of DNA in 154 mM NaCl at 4 degrees C. Neither turbidity nor precipitation was observed in PLL-g-HA/DNA mixtures over the copolymer (based on PLL) to DNA charge ratio from 0 to 2. This indicated that inter-polyelectrolyte complex formation between PLL-g-HA and DNA exhibited minimal self-aggregation, explaining the highly soluble nature of the complex. The agarose gel retardation assay revealed that the titration point representing the minimum proportion of PLL-g-HA required to retard the DNA completely occurred at a 1:1 copolymer to DNA charge ratio.Intravenous injection of the [P-32]pSV beta-Gal plasmid (an expression plasmid encoding lacZ) complexed to PLL-g-HA in Wistar rats demonstrated specific hepatic targeting with >93% of the injected counts taken up by the liver in 1 h. Further using an FITC-labeled DNA, it was shown that the PLL-g-HA/DNA complex was distributed exclusively in the SEC. Seventy-two hours after injection of 90 mu g of pSV beta-Gal in a PLL-g-HA-complexed form, a large number of SEC expressing beta-galactosidase were detected. The new PLL-g-HA/DNA carrier system permits targeted delivery of exogenous genes selectively to the liver SEC.

Mitochondria are frequently the target of injury after stresses leading to necrotic and apoptotic cell death. Inhibition of oxidative phosphorylation progresses to uncoupling when opening of a high conductance permeability transition (PT) pore in the mitochondrial inner membrane abruptly increases the permeability of the mitochondrial inner membrane to solutes of molecular mass up to 1500 Da. Cyclosporin A (CsA) blocks this mitochondrial permeability transition (MPT) and prevents necrotic cell death from oxidative stress, Ca2+ ionophore toxicity, Reye related drug toxicity, pH-dependent ischemia/reperfusion injury, and other models of cell injury. Confocal fluorescence microscopy directly visualizes onset of the MPT from the movement of green-fluorescing calcein into mitochondria and the simultaneous release from mitochondria of red-fluorescing tetramethylrhodamine methylester, a membrane potential-indicating fluorophore. In oxidative stress to hepatocytes induced by tert-butylhydroperoxide, NAD(P)H oxidation, increased mitochondrial Ca2+, and mitochondrial generation of reactive oxygen species precede and contribute to onset of the MPT. Confocal microscopy also shows directly that the MPT is a critical event in apoptosis of hepatocytes induced by tumor necrosis factor-alpha. Progression to necrotic and apoptotic cell killing depends, at least in part, on the effect the MPT has on cellular ATP levels. If ATP levels fall profoundly, necrotic killing ensues. If ATP levels are at least partially maintained, apoptosis follows the MPT. Cellular features of both apoptosis and necrosis frequently occur together:after death signals and toxic stresses. A new term, necrapoptosis, describes such death processes that begin with a common stress or death signal, progress by shared pathways, but Culminate in either cell lysis (necrosis) or programmed cellular resorption (apoptosis) depending on modifying factors such as ATP.

Onset of the cyclosporin-A-sensitive mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by laser scanning confocal microscopy. The MPT is a causative event in many types of necrotic and apoptotic cell death, including oxidative stress, ischemia/reperfusion injury, Ca2+ ionophore toxicity and tumor necrosis factor alpha (TNF alpha) induced apoptosis, and may contribute to Reye's-related drug toxicity. Pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and increased mitochondrial Ca2+ and pH can each promote onset of the MPT in situ. The MPT can also be directly visualized during TNF alpha-induced apoptosis to hepatocytes. Mitochondria spontaneously depolarize in situ after nutrient deprivation before entering an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. We propose a model in which onset of the MPT to increasing numbers of mitochondria leads progressively to autophagy, apoptosis and necrotic cell death.

Using confocal microscopy, onset of the mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by the redistribution of the cytosolic fluorophore, calcein, into mitochondria. Simultaneously, mitochondria release membrane potential-indicating fluorophores like tetramethylrhodamine methylester. The MPT occurs in several forms of necrotic cell death, including oxidative stress, pH-dependent ischemia/reperfusion injury and Ca2+ ionophore toxicity. Cyclosporin A (CsA) and trifluoperazine block the MPT in these models and prevent cell killing, showing that the MPT is a causative factor in necrotic cell death. During oxidative injury induced by t-butylhydroperoxide, onset of the MPT is preceded by pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and an increase of mitochondrial free Ca2+, all changes that promote the MPT. During tissue ischemia, acidosis develops. Because of acidotic pH, anoxic cell death is substantially delayed. However, when pH is restored to normal after reperfusion (reoxygenation at pH 7.4), cell death occurs rapidly (pH paradox). This killing is caused by pH-dependent onset of the MPT, which is blocked by reperfusion at acidotic pH or with CsA. In isolated mitochondria, toxicants causing Reye's syndrome, such as salicylate and valproate, induce the MPT. Similarly, salicylate induces a CsA-sensitive MPT and killing of cultured hepatocytes. These in vitro findings suggest that the MPT is the pathophysiological mechanism underlying Reye's syndrome in vivo. Kroemer and coworkers proposed that the MPT is a critical event in the progression of apoptotic cell death. Using confocal microscopy, the MPT can be directly documented during tumor necrosis factor-alpha induced apoptosis in hepatocytes. CsA blocks this MPT and prevents apoptosis. The MPT does not occur uniformly during apoptosis. Initially, a small proportion of mitochondria undergo the MPT, which increases to nearly 100% over 1-3 h. A technique based on fluorescence resonance energy transfer can selectively reveal mitochondrial depolarization. After nutrient deprivation, a small fraction of mitochondria spontaneously depolarize and enter an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. A model is proposed in which onset of the MPT to increasing numbers of mitochondria within a cell leads progressively to autophagy, apoptosis and necrotic cell death.