Comparison of effect of selected synthetic chalcone analogues on mitochondrial outer membrane determined by fluorescence spectroscopy

Effect on mitochondrial outer membrane of six selected synthetic cyclic chalcone analogues, E-2-arylmethylene-1-tetralones (2) and E-2-arylmethylene-1-benzosuberones (3), were investigated by fluorescence spectroscopy. The selected compounds represent derivatives with different degree of cytotoxicity against murine and human cancer lines. Excitation and emission fluorescence spectra of the cyclic chalcone analogues 2 and 3 were recorded in respiration medium containing 1 mM succinate. It was found that the ring size as well as the nature and location of the aromatic substituents have significant effect on fluorescence of the compounds. Interaction of subtoxic concentration of compounds 2 and 3 with the outer mitochondrial membrane was investigated by recording their fluorescence polarization in the presence of rat liver mitochondria. The most cytotoxic E-2-(4'-methoxybenzylidene)-1-benzosuberone (3b) was found to display a continuous increase of fluorescence polarization signal in the presence of mitochondria--a different pattern of interaction with the mitochondrial outer membrane from that observed for rest of the investigated compounds.     

Assessment of the effect of cyclic chalcone analogues on mitochondrial membrane and DNA

The cytotoxic and protective effects of selected synthetic chalcone analogues have been shown in previous studies. We studied their cytotoxic effect on the modification of mitochondrial membrane potential and on DNA. The first spectral information about the methoxy group as well as the dimethylamino substituent in E-2-arylmethylene-1-benzosuberones molecule was obtained by absorption and emission spectra. The cytotoxic effect of both cyclic chalcone analogues on DNA were detected by alkaline single-cell gel electrophoresis. Better fluorescent chalcone analogue E-2-(4′-dimethylamino-benzylidene)-1-benzosuberone was studied further in fresh isolated mitochondria. The decrease of rat liver mitochondria membrane potential (Δψ) was observed by fluorescence emission spectra. For the collapsing of mitochondrial potentials and as the negative control of mitochondrial function the CCCP uncoupler was used. The absorption maximum of the methoxy group was found at a shorter wavelength (λ = 335 nm) than that of the dimethylamino group (λ = 406 nm). The excitation spectra were very similar to the absorption spectra for both molecules but the emission spectra showed a better fluorescence for dimethylamino derivative. After the addition of E-2-(4′-dimethylamino-benzylidene)-1-benzosuberone to the intact mitochondria the decrease of mitochondrial membrane potential Δψ was observed by emisssion fluorescence spectra. Both cyclic chalcone analogues induced DNA damage, which was detected by alkaline comet assay. Mainly the apoptotic cells were detected, but necrotic cells were also present. Similarities in the percentages of DNA migration from the head were observed in both treatment groups. Both benzosuberones, with dimethylamino- and methoxy- substituent, were very active biologically, as shown by DNA results of the comet assay. Due to its better fluorescence properties, only the fluorophore with dimethylamino substituent was selected for further study of the function of rat liver mitochondria. Decline of mitochondrial function as well as mitochondrial DNA damage were evident between experimental and control groups.     

Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:coenzyme A (CoA) ligase (4CL) from Arabidopsis thaliana, and chalcone synthase (CHS) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans-cinnamic acid and p-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter(-1) of naringenin and 0.8 mg liter(-1) of pinocembrin. Several by-products, such as 2',4',6'-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor l-tyrosine.     

Nitric oxide and mitochondrial status in noise-induced hearing loss

The study investigated the distribution of nitric oxide (NO) within isolated outer hair cells (OHCs) from the cochlea, its relationship to mitochondria and its modulation of mitochondrial function. Using two fluorescent dyes--4,5-diamino-fluorescein diacetate (DAF-2DA), which detects NO, and tetramethyl rhodamine methyl ester (TMRM+), a mitochondrial membrane potential dye--it was found that a relatively greater amount of the DAF fluorescence in OHCs co-localized with mitochondria in comparison to DAF fluorescence in the cytosole. This study also observed reduced mitochondrial membrane potential of OHCs and increased DAF fluorescence following exposure of the cells to noise (120 dB SPL for 4 h) and to an exogenous NO donor, NOC-7 (>350 mm). Antibody label for nitrotyrosine was also increased, indicating NO-related formation of peroxynitrite in both mitochondria and the cytosol. The results suggest that NO may play an important physiological role in regulating OHC energy status and act as a potential agent in OHC pathology.     

Inhibitory effect of naringenin chalcone on inflammatory changes in the interaction between adipocytes and macrophages

Obese adipose tissue is characterized by an enhanced infiltration of macrophages. It is considered that the paracrine loop involving monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-alpha between adipocytes and macrophages establishes a vicious cycle that augments the inflammatory changes and insulin resistance in obese adipose tissue. Polyphenols, which are widely distributed in fruit and vegetables, can act as antioxidants and some of them are also reported to have anti-inflammatory properties. Tomato is one of the most popular and extensively consumed vegetable crops worldwide, which also contains many flavonoids, mainly naringenin chalcone. We investigated the effect of flavonoids, including naringenin chalcone, on the production of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated macrophages and in the interaction between adipocytes and macrophages. Naringenin chalcone inhibited the production of TNF-alpha, MCP-1, and nitric oxide (NO) by LPS-stimulated RAW 264 macrophages in a dose-dependent manner. Coculture of 3T3-L1 adipocytes and RAW 264 macrophages markedly enhanced the production of TNF-alpha, MCP-1, and NO compared with the control cultures; however, treatment with naringenin chalcone dose-dependently inhibited the production of these proinflammatory mediators. These results indicate that naringenin chalcone exhibits anti-inflammatory properties by inhibiting the production of proinflammatory cytokines in the interaction between adipocytes and macrophages. Naringenin chalcone may be useful for ameliorating the inflammatory changes in obese adipose tissue.     

Anti-allergic activity of naringenin chalcone from a tomato skin extract

The anti-allergic activity of a tomato extract was studied by using an in vitro histamine-release assay. The tomato skin extract exerted the strongest inhibition of histamine release. Chlorogenic acid, rutin and naringenin were identified in the 60% ethanol extract of tomato skin. However, the extract contained an unknown compound which strongly inhibited histamine release. This active compound in tomato skin was identified as naringenin chalcone (trans-2'4'6'4-tetrahydroxychalcone). Naringenin chalcone inhibited histamine release with an IC(50) value of 68 microg/ml. The anti-allergic activity of the tomato skin extract was next investigated by the in vivo mouse ear-swelling response. We found that naringenin chalcone showed the strongest inhibitory effect of the polyphenols of the tomato skin extract. These results indicate that a tomato skin extract could inhibit allergic reactions.     

Estimation of the mitochondrial calcium pool in rat brain synaptosomes using Rhod-2 AM fluorescent dye

The literature data on the role of synaptic mitochondria in the regulation of the cytosolic calcium level are contradictory. In the present paper calcium storage by mitochondria in rat brain synaptosomes using the fluorescent dye Rhod-2 has been investigated. The addition of 60 mM KCl increases Rhod-2 fluorescence. This effect is completely abolished by replacing K⁺ with Na⁺ or withdrawing Ca²⁺ from the incubation medium. A proton ionophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone, and a mixture of rotenone/oligomycin mitochondrial toxins cause a two-fold decrease in Rhod-2 fluorescence. Thapsigargin, an inhibitor of endoplasmic reticulum ATPase (1 μM), but not bafilomycin, an inhibitor of ATPase in synaptic vesicles (500 nM) also leads to a mitochondrial calcium influx. The addition of calcium to synaptosomes with the retained plasma membrane potential increased Rhod-2 fluorescence; however, this effect is insensitive to carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. We have shown that mitochondria can serve as a calcium store in synaptosomes only in the case of a high cytosolic concentration of calcium.     

Phosphate is required to maintain the outer membrane integrity and membrane potential of respiring yeast mitochondria.

The buffer requirements to maintain mitochondrial intactness and membrane potential in in vitro studies were investigated, using gradient purified yeast mitochondria. It was found that the presence of phosphate is crucial for generation of a stable membrane potential and for preserving the intactness of the outer membrane, as assessed by probing the accessibility of Tom40p to trypsin and the leakage of cytochrome b2 from the intermembrane space. Upon addition of respiratory substrate in the absence of phosphate, mitochondria generate a membrane potential that collapses within 1 min. Under the same conditions, the mitochondrial outer membrane is disrupted. The presence of phosphate prevents both phenomena. The DeltapH component of the proton motive force appears to be responsible for the compromised outer membrane integrity. The collapse of the membrane potential is reversible to a limited extent. Only when phosphate is added soon enough after the addition of exogenous respiratory substrate can a stable membrane potential be obtained again. Within a few minutes, this capacity is lost. The presence of Mg(2+) prevents rupture of the outer membrane, but does not prevent rapid dissipation of the membrane potential. Similar results were obtained for mitochondria isolated and stored in the presence of dextran or bovine serum albumin.     

Flavonoids naringenin chalcone,naringenin, dihydrotricin,and tricin are lignin monomers in papyrus

Recent studies demonstrate that several polyphenolic compounds produced from beyond the canonical monolignol biosynthetic pathways can behave as lignin monomers, participating in radical coupling reactions and being incorporated into lignin polymers. Here, we show various classes of flavonoids, the chalconoid naringenin chalcone, the flavanones naringenin and dihydrotricin, and the flavone tricin, incorporated into the lignin polymer of papyrus (Cyperus papyrus L.) rind. These flavonoids were released from the rind lignin by Derivatization Followed by Reductive Cleavage (DFRC), a chemical degradative method that cleaves the β-ether linkages, indicating that at least a fraction of each was integrated into the lignin as β-ether-linked structures. Due to the particular structure of tricin and dihydrotricin, whose C-3ʹ and C-5ʹ positions at their B-rings are occupied by methoxy groups, these compounds can only be incorporated into the lignin through 4ʹ–O–β bonds. However, naringenin chalcone and naringenin have no substituents at these positions and can therefore form additional carbon–carbon linkages, including 3ʹ– or 5ʹ–β linkages that form phenylcoumaran structures not susceptible to cleavage by DFRC. Furthermore, Nuclear Magnetic Resonance analysis indicated that naringenin chalcone can also form additional linkages through its conjugated double bond. The discovery expands the range of flavonoids incorporated into natural lignins, further broadens the traditional definition of lignin, and enhances the premise that any phenolic compound present at the cell wall during lignification could be oxidized and potentially integrated into the lignin structure, depending only on its chemical compatibility. This study indicates that papyrus lignin has a unique structure, as it is the only lignin known to date that integrates such a diversity of phenolic compounds from different classes of flavonoids. This discovery will open up new ways to engineer and design lignins with specific properties and for enhanced value.

A series of flavonoids incorporate into the rind lignin of papyrus, participating as monomers during lignification.     

Reduction of membrane fluidity by antibacterial sophoraflavanone G isolated from Sophora exigua.

Sophoraflavanone G (5,7,2',4'-tetrahydroxy-8-lavandulylflavanone) has been referred as a phytochemical with the intensive antibacterial activity. To elucidate the pharmacological mechanism underlying an antibacterial action, sophoraflavanone G was isolated from Sophora exigua, thereafter its effect on membrane fluidity was studied using model membranes and compared with less active naringenin lacking 8-lavandulyl and 2'-hydroxyl groups. Highly purified sophoraflavanone G of 0.05-5 microg/ml, corresponding to the minimum growth inhibitory concentrations against various bacteria, significantly increased fluorescence polarization of the liposomes prepared from 1,2-dipalmitoyl-L-alpha-phosphatidylcholine and 1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine. Such increases were found in both liposomes measured with two fluorescent probes to indicate an alteration of membrane fluidity in hydrophilic and hydrophobic regions, suggesting that sophoraflavanone G reduces the fluidity of outer and inner layers of membranes. Although naringenin also showed the membrane effect, it needed concentrations over 2.5 microg/ml to induce a significant reduction of membrane fluidity. Sophoraflavanone G is considered to exert an antibacterial effect by reducing the fluidity of cellular membranes.     

Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol

Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O(2)*-) and hydrogen peroxide (H(2)O(2)). The electron transfer chain of mitochondria is a well documented source of H(2)O(2); however, the release of O(2)*- from mitochondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O(2)*-, elucidating the mechanisms underlying the release of O(2)*- from mitochondria into cytosol, and assessing the role of outer membrane voltage-dependent anion channels (VDACs) in this process. Isolated rat heart mitochondria supplemented with complex I or II substrates generate an EPR signal ascribed to O(2)*-. Inhibition of the signal in a concentration-dependent manner by both manganese-superoxide dismutase and cytochrome c proteins that cannot cross the mitochondrial membrane supports the extramitochondrial location of the spin adduct. Basal rates of O(2)*- release from mitochondria were estimated at approximately 0.04 nmol/min/mg protein, a value increased approximately 8-fold by the complex III inhibitor, antimycin A. These estimates, obtained by quantitative spin-trapping EPR, were confirmed by fluorescence techniques, mainly hydroethidine oxidation and horseradish peroxidase-based p-hydroxyphylacetate dimerization. Inhibitors of VDAC, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), and dextran sulfate (in a voltage-dependent manner) inhibited O(2)*- production from mitochondria by approximately 55%, thus suggesting that a large portion of O(2)*- exited mitochondria via these channels. These findings are discussed in terms of competitive decay pathways for O(2)*- in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments.     

Reduction of Brain Mitochondrial β-Oxidation Impairs Complex I and V in Chronic Alcohol Intake: The Underlying Mechanism for Neurodegeneration

Neuropathy and neurocognitive deficits are common among chronic alcohol users, which are believed to be associated with mitochondrial dysfunction in the brain. The specific type of brain mitochondrial respiratory chain complexes (mRCC) that are adversely affected by alcohol abuse has not been studied. Thus, we examined the alterations of mRCC in freshly isolated mitochondria from mice brain that were pair-fed the ethanol (4% v/v) and control liquid diets for 7–8 weeks. We observed that alcohol intake severely reduced the levels of complex I and V. A reduction in complex I was associated with a decrease in carnitine palmitoyltransferase 1 (cPT1) and cPT2 levels. The mitochondrial outer (cPT1) and inner (cPT2) membrane transporter enzymes are specialized in acylation of fatty acid from outer to inner membrane of mitochondria for ATP production. Thus, our results showed that alterations of cPT1 and cPT2 paralleled a decrease β-oxidation of palmitate and ATP production, suggesting that impairment of substrate entry step (complex I function) can cause a negative impact on ATP production (complex V function). Disruption of cPT1/cPT2 was accompanied by an increase in cytochrome C leakage, while reduction of complex I and V paralleled a decrease in depolarization of mitochondrial membrane potential (ΔΨ, monitored by JC-1 fluorescence) and ATP production in alcohol intake. We noted that acetyl-L-carnitine (ALC, a cofactor of cPT1 and cPT2) prevented the adverse effects of alcohol while coenzyme Q10 (CoQ10) was not very effective against alcohol insults. These results suggest that understanding the molecular, biochemical, and signaling mechanisms of the CNS mitochondrial β-oxidation such as ALC can mitigate alcohol related neurological disorders.     

Study of soluble lipoprotein in rat liver mitochondria

1. A water-soluble lipoprotein was isolated and purified from osmotically shocked preparations of rat liver mitochondria by using a technique of Sephadex-sandwich disc electrophoresis. 2. The purified lipoprotein migrates as a distinct sharp zone in high-resolution electrophoretic systems, indicating high degree of purity. 3. The lipoprotein resembles mitochondrial membranes with respect to lipid composition and lipid/protein ratio. 4. The lipoprotein and its apoprotein fraction obtained by delipidization at -18 degrees C to -20 degrees C have common properties with respect to their fluorescence spectra, instability to storage and electrophoretic mobility. 5. The purified lipoprotein has an excitation maximum at 325nm and a fluorescence maximum at 418nm. 6. Storage at 4 degrees C for 4 days or repeated freezing and thawing results in 15-30% decrease in electrophoretic mobility. 7. The patterns of incorporation in vitro of [1-(14)C]leucine into proteins of the soluble lipoprotein and of mitochondrial membrane of isolated rat liver mitochondria suggest a probable precursor role for the apoprotein in the formation of mitochondrial membrane protein. 8. Lipoprotein preparations isolated from mitochondrial fractions of rat kidney, brain and heart and of chicken and mouse liver resemble closely that obtained from rat liver mitochondria, suggesting that the soluble lipoprotein could be a distinct entity of mitochondrial origin.     

Mitochondrial degradation by autophagy (mitophagy) in GFP-LC3 transgenic hepatocytes during nutrient deprivation

Fasting in vivo and nutrient deprivation in vitro enhance sequestration of mitochondria and other organelles by autophagy for recycling of essential nutrients. Here our goal was to use a transgenic mouse strain expressing green fluorescent protein (GFP) fused to rat microtubule-associated protein-1 light chain 3 (LC3), a marker protein for autophagy, to characterize the dynamics of mitochondrial turnover by autophagy (mitophagy) in hepatocytes during nutrient deprivation. In complete growth medium, GFP-LC3 fluorescence was distributed diffusely in the cytosol and incorporated in mostly small (0.2-0.3 μm) patches in proximity to mitochondria, which likely represent preautophagic structures (PAS). After nutrient deprivation plus 1 μM glucagon to simulate fasting, PAS grew into green cups (phagophores) and then rings (autophagosomes) that enveloped individual mitochondria, a process that was blocked by 3-methyladenine. Autophagic sequestration of mitochondria took place in 6.5 ± 0.4 min and often occurred coordinately with mitochondrial fission. After ring formation and apparent sequestration, mitochondria depolarized in 11.8 ± 1.4 min, as indicated by loss of tetramethylrhodamine methylester fluorescence. After ring formation, LysoTracker Red uptake, a marker of acidification, occurred gradually, becoming fully evident at 9.9 ± 1.9 min of ring formation. After acidification, GFP-LC3 fluorescence dispersed. PicoGreen labeling of mitochondrial DNA (mtDNA) showed that mtDNA was also sequestered and degraded in autophagosomes. Overall, the results indicate that PAS serve as nucleation sites for mitophagy in hepatocytes during nutrient deprivation. After autophagosome formation, mitochondrial depolarization and vesicular acidification occur, and mitochondrial contents, including mtDNA, are degraded.     

外周苯二氮(艹卓)受体激动剂Ro5-4864抑制大鼠心肌细胞线粒体通透性转换

线粒体通透性转换（mitochondrial permeability transition,MPT）导致线粒体氧化应激性损伤。近年研究认为,位于线粒体外膜的外周苯二氮节受体（peripheral benzodiazepine receptor,PBR）参与了线粒体的重要生理功能。本研究在心肌细胞线粒体水平探讨激动PBR能否抑制Ca^2＋诱发的MPT。分离Sprague—Dawley大鼠心肌细胞线粒体,将PBR激动剂Ro5-4864（50、100、200μmol／L）和线粒体孵育,利用150μmol／L Ca^2＋诱发MPT,部分线粒体在与100μmol／L Ro5-4864孵育前5min加入MPT孔道开放剂苍术苷（atractyloside,ATR）。采用分光光度法观察线粒体膨胀情况：Westernblot检测线粒体细胞色素C（cytochrome C,CytoC）释放;利用荧光探针JC-1在激光共聚集显微镜下观察线粒体膜电位的变化。50、100、200μmol／L Ro5-4864均显著抑制Ca^2＋诱发的520nm处线粒体吸光度的下降,而且抑制Ca^2＋引起的线粒体CytoC释放和线粒体膜电位下降,但ATR可阻断R05—4864的上述作用。结果提示,PBR激动剂可抑制大鼠心肌MPT,保持线粒体CytoC含量和稳定线粒体膜电位,减轻线粒体损伤。PBR的激活可能成为减轻心肌细胞应激性损伤及心肌保护的新方法。     

Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals,enhancing cytochrome c release to the cytosol

Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.     

Use of Human Cancer Cell Lines Mitochondria to Explore the Mechanisms of BH3 Peptides and ABT-737-Induced Mitochondrial Membrane Permeabilization

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.     

NADH fluorescence of isolated ventricular myocytes: effects of pacing, myoglobin, and oxygen supply.

Endogenous fluorescence was used to measure the extent of reduction of mitochondrial NAD in individual, isolated rat cardiac myocytes. NAD reduction was determined from emitted fluorescence at 415 and 470 nm during brief epi-illumination at 365 nm. NAD reduction of resting myocytes, superfused with medium equilibrated with 95% O2/5% CO2, was 27 +/- 3% (SE) (n = 78), comparable to that in beating whole heart. Increasing intracellular Ca2+ did not significantly change NAD reduction. NAD reduction decreased reversibly to 11 +/- 1% (n = 78) in contracting myocytes electrically paced at 5 Hz for 10 min. Oxygen uptake was stimulated fivefold. There was minimal change in sarcoplasmic pH measured by fluorescence of carboxy-seminaphthorhodafluor-1. However, NAD reduction increased reversibly in response to electrically paced contractions when: (a) myoglobin was inactivated with sodium nitrite (37 +/- 7%; n = 48); or (b) cells were more densely layered and gassed with 20% O2/5% CO2 (48 +/- 3%; n = 30). We conclude that (a) the ratio NADH/NAD is decreased in well-oxygenated cells with increased work; (b) steady-state NAD reduction is increased with increased work when oxygen delivery is limited; and (c) functional myoglobin ensures an oxygen supply to the mitochondria of working cells.     

Biotransformation of naringin and naringenin by cultured Eucalyptus perriniana cells

The biotransformation of naringin and naringenin was investigated using cultured cells of Eucalyptus perriniana. Naringin (1) was converted into naringenin 7-O-β-d-glucopyranoside (2, 15%), naringenin (3, 1%), naringenin 5,7-O-β-d-diglucopyranoside (4, 15%), naringenin 4′,7-O-β-d-diglucopyranoside (5, 26%), naringenin 7-O-[6-O-(β-d-glucopyranosyl)]-β-d-glucopyranoside (6, β-gentiobioside, 5%), naringenin 7-O-[6-O-(α-l-rhamnopyranosyl)]-β-d-glucopyranoside (7, β-rutinoside, 3%), and 7-O-β-d-gentiobiosyl-4′-O-β-d-glucopyranosylnaringenin (8, 1%) by cultured cells of E. perriniana. On the other hand, 2 (14%), 4 (7%), 5 (13%), 6 (2%), 7 (1%), naringenin 4′-O-β-d-glucopyranoside (9, 4%), naringenin 5-O-β-d-glucopyranoside (10, 2%), and naringenin 4′,5-O-β-d-diglucopyranoside (11, 5%) were isolated from cultured E. perriniana cells, that had been treated with naringenin (3). Products, 7-O-β-d-gentiobiosyl-4′-O-β-d-glucopyranosylnaringenin (8) and naringenin 4′,5-O-β-d-diglucopyranoside (11), were hitherto unknown.     

The structure of the carbohydrate backbone of the lipopolysaccharide of Pectinatus frisingensis strain VTT E-79104

The structure of the carbohydrate backbone of the lipopolysaccharide from Pectinatus frisingensis strain VTT E-79104 was analyzed using chemical degradations, NMR spectroscopy, mass spectrometry, and chemical methods. The LPS contains two major structural variants, differing in the presence or absence of an octasaccharide fragment. The largest structure of the carbohydrate backbone of the LPS, that could be deduced from experimental results, consists of 20 monosaccharides arranged in a nonrepetitive sequence: [carbohydrate structure: see text] where R is H or 4-O-Me-alpha-L-Fuc-(1-2)-4-O-Me-beta-Hep-(1-3)-alpha-GlcNAc-(1-2)-beta-Man-(1-3)-beta-ManNAc-(1-4)-alpha-Gal-(1-4)-beta-Hep-(1-3)-beta-GalNAc-(1- where Hep is a residue of D-glycero-D-galacto-heptose; all monosaccharides have the D-configuration except for 4-O-Me-L-Fuc and L-Ara4N. This structure is architecturally similar to the oligosaccharide system reported previously in P. frisingensis VTT E-82164 LPS, but differs from the latter in composition and also in the size of the outer region.