Membrane Intactness Research Articles

Structural changes in the neocortex nervous tissue in rat ontogenesis after hypoxia at various terms of embryogenesis

The performed study has shown that in rats submitted to hypoxia (3 h, 7% O2) at the 14th day of embryogenesis (E14) as compared with control animals, density of distribution of cells in the brain cortex decreased for the first month of postnatal ontogenesis (maximally by 40.8% by P20). In dying neurons, swelling of the cell body, lyses of or ganoids, and disturbance of the cytoplasm membrane intactness were observed. Two waves of neuronal death by the mechanism of capsize-dependent apoptosis were revealed; the first involved large pyramidal neurons of the layer V (P10–20), the second-small pyramidal and non-pyramidal neurons of the layers II–III (P20–30). In neurosis of molecular layer, a decrease of the mean amount of labile synaptopodin-positive dendrite spines was observed, as compared with control. In rats exposed to hypoxia at E18, no changes of cell composition and structure of the nervous tissue were found in the studied brain cortex areas. Thus, formation of the cortex nervous tissue in postnatal ontogenesis of rats submitted to hypoxia at the period of neuroblast proliferation-migration is accompanied not only by a change of the cell composition of various cortex layers in early ontogenesis, but also by a decrease of the number of the synaptopodin-positive spines in the molecular layer, the decrease being preserved in adult animals.

Aluminum toxicity and Ca depletion may enhance cell death of tobacco cells via similar syndrome

The main objective of this work is to find out whether aluminum (Al) toxicity and Ca depletion cause cell death of tobacco cells via similar sequence of events. Tobacco cell suspension culture exhibited maximum fresh weight in the presence of a wide range of Ca concentrations between 0.1-1.0 mM whereas higher concentrations (> 1.0-5.0 mM) gradually lowered cell fresh weight. However, this decrease in fresh weight does not imply a negative impact on cell viability since cell growth recommenced in fresh MS medium with rates mostly higher than those of low Ca. In addition, high Ca seems to be crucial for survival of Al-treated cells. On the other side, tobacco cells exhibited extreme sensitivity to complete deprivation of Ca. Without Ca, cells could not survive for 18 h and substantially lost their growth capability. Evans blue uptake proved membrane damage of Ca-depleted same as Al-treated cells; relative to maintained membrane intactness of calcium-supplemented (control) ones. Percentage of membrane damage and the growth capability (survival) of tobacco cells exhibited a clear negative correlation. Alterations in growth (fresh weight per aliquot) could not be ascribed neither to cell number nor to decreased dry matter allocation (dry weight/fresh weight percentage) but was mainly due to decreased cellular water content. In this context, Ca-depleted cells lost about half their original water content while 100 μM Al-treated ones retained most of it (ca 87%). This represented the single difference between the two treatments (discussed in the text). Nevertheless, such high water content of the Al-treated cells seems physiologically useless since it did not result in improved viability. Similarities, however, included negligible levels of growth capability, maximum levels of membrane damage, and comparable amounts of NO3- efflux. As well, both types of treatments led to a sharp decline in osmotic potential that is, in turn, needed for water influx. The above-mentioned sequence of events, induced by Al application looks, to a great extent, similar to Ca depletion syndrome leading finally to cell death of tobacco cells.

Functional Sieve Element Protoplasts

Sieve element (SE) protoplasts were liberated by exposing excised phloem strands of Vicia faba to cell wall-degrading enzyme mixtures. Two types of SE protoplasts were found: simple protoplasts with forisome inclusions and composite twin protoplasts-two protoplasts intermitted by a sieve plate-of which one protoplast often includes a forisome. Forisomes are giant protein inclusions of SEs in Fabaceae. Membrane integrity of SE protoplasts was tested by application of CFDA, which was sequestered in the form of carboxyfluorescein. Further evidence for membrane intactness was provided by swelling of SE protoplasts and forisome dispersion in reaction to abrupt lowering of medium osmolarity. The absence of cell wall remnants as demonstrated by negative Calcofluor White staining allowed patch-clamp studies. At negative membrane voltages, the current-voltage relations of the SE protoplasts were dominated by a weak inward-rectifying potassium channel that was active at physiological membrane voltages of the SE plasma membrane. This channel had electrical properties that are reminiscent of those of the AKT2/3 channel family, localized in phloem cells of Arabidopsis (Arabidopsis thaliana). All in all, SE protoplasts promise to be a powerful tool in studying the membrane biology of SEs with inherent implications for the understanding of long-distance transport and signaling.

Molecular and Biochemical Analysis of the Plastidic ADP-glucose Transporter (ZmBT1) from Zea mays

Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous synthesis of ZmBT1 in Escherichia coli cells leads to the functional integration of ZmBT1 into the bacterial cytoplasmic membrane. ZmBT1 transports ADP-Glc in counterexchange with ADP with apparent affinities of about 850 and 465 mum, respectively. Recently, a complete ferredoxin/thioredoxin system has been identified in cereal amyloplasts and BT1 has been proposed as a potential Trx target protein (Balmer, Y., Vensel, W. H., Cai, N., Manieri, W., Schurmann, P., Hurkman, W. J., and Buchanan, B. B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 2988-2993). Interestingly, we revealed that the transport activity of ZmBT1 is reversibly regulated by redox reagents such as diamide and dithiothreitol. The expression of ZmBT1 is restricted to endosperm tissues during starch synthesis, whereas a recently identified BT1 maize homologue, the ZmBT1-2, exhibits a ubiquitous expression pattern in hetero- and autotrophic tissues indicating different physiological roles for both maize BT1 isoforms. BT1 homologues are present in both mono- and dicotyledonous plants. Phylogenetic analyses classify the BT1 family into two phylogenetically and biochemically distinct groups. The first group comprises BT1 orthologues restricted to cereals where they mediate the ADP-Glc transport into cereal endosperm storage plastids during starch synthesis. The second group occurs in mono- and dicotyledonous plants and is most probably involved in the export of adenine nucleotides synthesized inside plastids.

Seasonality affects post-thaw plasma membrane intactness and sperm velocities in spermatozoa from Thai AI swamp buffaloes ( Bubalus bubalis)

Altogether 218 frozen semen AI doses, prepared between 1980 and 1989 and also between 2003 and 2005 from 18 AI Thai swamp buffalo sires, were examined to determine whether seasonality affects post-thaw viability, as plasma membrane integrity (PMI, using SYBR-14/PI), plasma membrane stability (PMS, using Annexin-V/PI), or motility (Mot, using CASA). A thermoresistance test (38 °C for 60 min) was used to further analyze sperm survivability in vitro. All variables were compared over 3 seasons of the year (rainy: July–October; winter: November–February; and summer: March–June), with distinct ambient temperature and humidity. PMI (% of alive spermatozoa) was higher in winter (54.6%, P < 0.001) than in the rainy (43.5%) or summer (46.7%) seasons. Outcomes of PMS (Annexin-V/PI assay) confirmed those of PMI, the highest PMS in spermatozoa processed in winter (55.7%, P < 0.001). Spermatozoa depicting linear Mot post-thaw ranged from 48.2% to 48.8% across seasons (ns), proportions that decreased during incubation (33.5–37.9%), albeit without seasonal differences. The mean percentages of straight linear velocity (VSL), average path velocity (VAP), or curvilinear velocity (VCL) were higher ( P < 0.05–0.001) in the rainy season than in winter or summer, while average lateral head displacement (ALH) was higher ( P < 0.05) in summer, differences maintained after incubation. In conclusion, post-thaw PMS and PMI, assessed by flow cytometry, were significantly better in sperm samples processed during winter than in samples processed during the other seasons of the year, a seasonal difference not picked up by CASA, probably due to the larger number of spermatozoa assessed.

Patterns of SPARC expression and basement membrane intactness at the tumour–brain border of invasive meningiomas

The matricellular glycoprotein SPARC (secreted protein, acidic and rich in cysteine), also termed osteonectin, has been found to regulate the invasive behaviour of several tumour types by interacting with basement membrane constituents. Brain invasive meningiomas are supposed to disrupt the pial-glial basement membrane. In the present study we aimed at determining the relationship of basement membrane intactness and SPARC protein expression at the meningioma-brain border. Sections of 51 brain-invasive meningiomas (31 meningothelial meningiomas WHO grade I, 11 atypical WHO grade II, and nine anaplastic WHO grade III tumours) were immunolabelled with antibodies against SPARC, epithelial membrane antigen (EMA), collagen IV and glial fibrillary acidic protein (GFAP). Twenty-two non-invasive WHO grade I meningothelial meningiomas were included in the study for comparison. At the tumour-brain border of invasive meningiomas, spindle-shaped tumour cells expressed SPARC. The number of tumours containing SPARC+ spindle cells did not differ significantly between WHO grades. By contrast, the number of WHO grade I tumours expressing collagen IV (15/31) was highly significantly elevated when compared with WHO grade II (1/11) and WHO grade III (0/9) (both P < 0.0001). There was an inverse relationship of the presence of SPARC+ spindle cells and basement membrane material. In conclusion, the destruction of the basement membrane is correlated with meningioma malignancy grade whereas the expression of SPARC protein at the tumour-brain border is not. Destruction of the basement membrane and appearance of SPARC+ spindle cells are not coincident during the course of brain invasion by meningiomas.

Detection of early changes in sperm membrane integrity pre-freezing can estimate post-thaw quality of boar spermatozoa

A recently developed triple staining (SNARF-1/YO-PRO-1/ethidium homodimer) was used to assess early changes in boar sperm membrane integrity (MI) with the results of cryopreservation procedures and to seek for correlations among MI-spermatozoa in pre-freeze semen and its freezeability. Ejaculates from five boars were evaluated in the fresh and frozen-thawed (FT) state, and its freezeability defined as % of membrane intactness, MI% (MI% = % of FT-spermatozoa with intact membranes × 100 divided by the % of pre-freeze spermatozoa with intact membranes) estimated. Significant differences were found among boars for freezeability (MI%) and motility post-thaw (%). Interestingly, significant correlations were found between the percentage of YO-PRO-1-positive spermatozoa and freezeability ( R = 0.440, p < 0.01), indicating this new triple staining can be used to safely disclose among ejaculates prior to freezing.

Dequalinium-induced Protofibril Formation of α-Synuclein

alpha-Synuclein is the major constituent of Lewy bodies, a pathological signature of Parkinson disease, found in the degenerating dopaminergic neurons of the substantia nigra pars compacta. Amyloidosis generating the insoluble fibrillar protein deposition has been considered to be responsible for the cell death observed in the neurodegenerative disorder. In order to develop a controlling strategy toward the amyloid formation, 1,1'-(1,10-decanediyl)-bis-[4-a-mino-2-methylquinolinium] (dequalinium), was selected and examined in terms of its specific molecular interaction with alpha-synuclein. The protein was self-oligomerized by dequalinium, which gave rise to the ladder formation on N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine/SDS-PAGE in the presence of a coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. The double-headed structure of dequalinium with the two cationic 4-aminoquinaldinium rings was demonstrated to be critical for the protein self-oligomerization. The dequalinium-binding site was located on the acidic C-terminal region of the protein with an approximate dissociation constant of 5.5 mum. The protein self-oligomerization induced by the compound has resulted in the protofibril formation of alpha-synuclein before it has developed into amyloids. The protofibrils were demonstrated to affect the membrane intactness of liposomes, and they have also been shown to influence cell viability of human neuroblastoma cells. In addition, dequalinium treatment of the alpha-synuclein-overexpressing cells exerted a significant cell death. Therefore, it is pertinent to consider that dequalinium could be used as a molecular probe to assess toxic mechanisms related to the amyloid formation of alpha-synuclein. Ultimately, the compound could be employed to develop therapeutic and preventive strategies toward alpha-synucleinopathies including Parkinson disease.

The Role of Mercury in the Etiology of Sperm Dysfunction in Holstein Bulls

A large number of toxicological substances and pharmacological and physical agents can cause reproductive intervention at the cellular and molecular level. The present study was designed to assess the effect of mercury (HgCl2) at 50 to 550 µM concentration ranges, in vitro, on the sperm membrane and DNA integrity, viability, and acrosomal status of normal bull spermatozoa. The samples were processed for sperm analyses using semen-diluting fluid (PBS, pH 7.2). We recorded a sharp increase in the lipid peroxidation/LPO rate; the highest was at 550 µM mercury concentration, indicating a deleterious effect of mercury on the sperm membrane intactness. There was also a strong negative correlation between LPO rate and % viable spermatozoa (R = 0.987, p<0.001). Data obtained from a comet assay technique revealed that mercury is capable of inducing DNA breaks in the sperm nuclei. Interestingly, 92% of DNA breaks were double-stranded. The correlation between LPO rate and % DNA breaks was 0.984. Performing the gelatin test indicates that mercury is able to alter the integrity of acrosomal membranes showing an abnormal acrosome reaction. In this regard, a strong link was found between LPO rate and % halos (R = 0.990, p<0.001). Collectively, mercury proved to be a potent oxidant in the category of environmental factors affecting bull spermatozoa. Hence, considering the wide spread use of mercury and its compounds, these metals should be regarded with more concern. (Asian-Aust. J. Anim. Sci. 2006. Vol 19, No. 3 : 335-340)

Bull Spermatozoa under Mercury Stress

Defective sperm function is the most prevalent cause of male infertility and is difficult to treat. This study was designed to evaluate the effect of mercuric chloride (HgCl2) at 50-300 micromol/l concentration range, in vitro, on the sperm membrane and DNA integrity, viability, reduced glutathione (GSH) content and acrosomal status of the bull spermatozoa. The samples were processed for sperm analyses using semen-diluting fluid [phosphate-buffered saline (PBS), pH 7.2]. I recorded a meaningful increase in the lipid peroxidation (LPO) rate and a drastic fall in the spermatocrit values under mercury additions, dominantly at 300 microM mercury concentration, indicating a deleterious effect of mercury on the sperm membrane intactness. There was also a strong negative correlation between LPO rate and percentage of viable spermatozoa (r = -0.9, p < 0.001). GSH content was significantly impaired. Data obtained from Comet assay [single-cell gel electrophoresis (SCGE)] technique revealed that mercury is capable of inducing DNA breaks in the sperm nuclei. Interestingly, 90% of DNA breaks were double-stranded. The correlation between LPO rate and percentage of DNA breaks was found to be 0.9 (p < 0.001). Results of the gelatin test indicate that mercury is capable of altering the integrity of acrosomal membranes, showing an abnormal acrosome reaction. In this regard, a strong correlation was found between LPO rate and percentage of halos (r = -0.9, p < 0.001). In conclusion, mercury proved to be a potential oxidant in the category of 'environmental factors' to bull spermatozoa. Hence, considering the widespread use of mercury and its compounds, these metals should be regarded with more concern.

Nicotinic infertility: assessing DNA and plasma membrane integrity of human spermatozoa.

Infertility remains a major problem in society, with recent data suggesting its presence in one of four couples. The objective of the present study was to evaluate the impact of nicotine (0.25, 0.5 and 0.75 mm), as a major component of cigarette smoke, in vitro, on sperm membrane [by spermatocrit and lipoperoxidation (LPO) tests], DNA integrity (by Comet assay), and viability of spermatozoa (by eosin staining) from normozoospermic men. Sperm samples were washed and diluted with phosphate-buffered saline. A drop in spermatocrit values and an increase in thiobarbituric acid-reactive substances/LPO rate was observed with the addition of nicotine, predominantly at a concentration of 0.75 mm, indicating a deleterious effect of nicotine on sperm membrane intactness. There was also a strong negative correlation between LPO rate and percentage viable sperm cell (r = -0.990). Data obtained from Comet assay technique revealed that nicotine could induce double-stranded DNA breaks (11% in 0.75 mm concentration) in the sperm nuclei. The value of r between LPO rate and percentage Comets was found to be +0.976. Taken together, nicotine proved to be a potential oxidant agent in the category of environmental factors to the integrity of sperm plasma membrane and DNA.

Impact of oxygen stress and energy availability on membrane stability of plant cells.

This article reviews the relationship between the energy status of plant cells under O(2) stress (e.g. waterlogging) and the maintenance of membrane intactness, using information largely derived from suspension cultures of anoxia-intolerant potato cells. Energy-related parameters measured were fermentation end-products (ethanol, lactate, alanine), respiratory rate, ATP, adenylate energy charge, nitrate reductase activity and biomass. ATP synthesis rates were calculated from the first four parameters. Reactive oxygen species were estimated from H(2)O(2) and superoxide levels, and the enzymatic detoxification potential from the activity levels of catalase and superoxide dismutase. Structure-related parameters were total fatty acids, free fatty acids (FFAs), lipid hydroperoxides, total phospholipids, N-acylphosphatidylethanolamine (NAPE) and cell viability. The following issues are addressed in this review: (1) what is the impact of anoxia on membrane lipids and how does this relate to energy status; (2) does O(2) per se play a role in these changes; (3) under which conditions and to what extent does lipid peroxidation occur upon re-aeration; and (4) can the effects of re-aeration be distinguished from those of anoxia? The emerging picture is a reappraisal of the relative contributions of anoxia and re-aeration. Two successive phases (pre-lytic and lytic) characterize potato cells under anoxia. They are connected by a threshold in ATP production rate, below which membrane lipids are hydrolysed to FFAs, and NAPE increases. Since lipid peroxidation occurs only when cells are reoxygenated during the lytic phase, its biological relevance in an already damaged system is questionable.

Field fertility with exported boar semen frozen in the new FlatPack container

The present study tested the field fertility of frozen–thawed (FT) Swedish boar semen packaged in flat plastic containers (FlatPacks) and exported for artificial insemination (AI) to overseas nucleus herds. Semen from 47 Swedish boars of Landrace (L), Yorkshire (Y), and Hampshire (H) breeds was frozen using a lactose–egg yolk-based extender with 3% glycerol and 10 9 spermatozoa/ml in 5 ml FlatPacks. For all breeds, FT sperm membrane intactness averaged 60%, while mean FT sperm motility ranged from 49 to 53%. A total of 308 litters resulted from 421 overseas inseminations with FT semen, with a mean farrowing rate (FR) of 73% and 10.7 mean number total piglets born. In a within-sow analysis for the purebred L and Y breedings, the FR and litter size of FT semen were compared with natural matings (NM) and on-farm AI with liquid semen (NM/AI breedings) at the same farms. Farrowing rate was 72.3 and 78.8% ( P=0.23), total piglets 11.3 and 11.6 ( P=0.44), and live piglets 10.1 and 10.2 ( P=0.77), for the FT semen and NM/AI breedings, respectively. The present results suggest that this freezing protocol and FlatPack container maintains high sperm viability post-thaw. Further the fertility levels when inseminated at overseas nucleus herds seem to be similar to those achieved with (NM/AI breedings) at the same farms. This freezing method may be a reliable alternative for the freezing/thawing of boar semen under commercial AI conditions.

Impaired cardiac mitochondrial membrane potential and respiration in copper-deficient rats.

Cardiac mitochondrial respiration, ATP synthase activity, and membrane potential and intactness were evaluated in copper-deficient rats. In the presence of NADH, both copper-deficient and copper-adequate mitochondria had very low oxygen consumption rates, indicating membrane intactness. However copper-deficient mitochondria had significantly lower oxygen consumption rates with NADH than did copper-adequate mitochondria. Copper-deficient mitochondria had significantly lower membrane potential than did copper-adequate mitochondria using fluorescent dyes. Copper-deficient mitochondria had significantly lower state 3 oxygen consumption rates and were less sensitive to inhibition by oligomycin, an ATP synthase inhibitor. Copper-deficient and copper-adequate mitochondria responded similiarly to CCCP. No difference was observed in mitochondrial ATPase activity between copper-deficient and copper-adequate rats using submitochondrial particles. We conclude that cardiac mitochondrial respiration is compromised in copper-deficient rats, and may be related to an altered ATP synthase complex and/or a decreased mitochondrial membrane potential.

Electrophysiological and biochemical changes in rabbit hearts stored at 4 C for 6 or 24 h

This study examines the electrophysiological and metabolic changes that occur in rabbit hearts during hypothermic storage in vitro. Hearts were microperfused at 4°C for 6 or 24h with either normal Krebs-Henseleit buffer (KHB) or KHB containing 2,3-butanedione monoxime (BDM). After hypothermic storage, hearts were rewarmed to 37°C with KHB. Cardiac function was then assessed in Langendorff perfusion mode. Electrophysiological changes were also assessed from the ventricular paced-evoked responses. After storage, mitochondria were isolated from the hearts and their respiratory control ratio, rate of ATP synthesis and outer membrane intactness were assessed. Compared with values from fresh non-stored hearts, hearts stored hypothermically for 24h showed significant decreases in both left ventricular developed pressure and coronary flow when reperfused in Langendorff mode. On the other hand, the decrease in left ventricular developed pressure in hearts that were stored for only 6h (with or without BDM) was not significant. Compared with values obtained from fresh non-stored hearts, hypothermic storage significantly decreased the R-wave amplitude, and both the R-E and ST-E intervals of paced-evoked responses. This was true for hearts microperfused for 6h (with or without BDM) and for hearts microperfused with buffer containing BDM for 24h. The ST-R intervals in hearts microperfused hypothermically for 6h were prolonged, but this change was not statistically significant compared with those obtained from unstored hearts. In hearts microperfused with KHB containing BDM for 24h, the ST-R interval was significantly prolonged. Hypothermic microperfusion for 24h significantly decreased both the mitochondrial coupling ratio and the rate of ATP synthesis. In hearts microperfused with BDM for 6h, mitochondrial coupling ratios and the rate of ATP synthesis were not significantly different from those in fresh hearts. In conclusion, the present study has shown that long-term hypothermic storage significantly impaired both paced-evoked responses and mitochondrial function. Inclusion of BDM in the perfusion buffer during storage significantly ameliorated some of these changes.

The plasma membrane intactness of root-tip cells is a primary factor for Al-tolerance in cultivars of five species

We investigated the role of the cell wall and plasma membrane (PM) of root-tip cells in Al tolerance in Al-tolerant and Al-sensitive cultivars of five plant species (rice, maize, pea, wheat, and sorghum). No correlation was found between the differences in Al tolerance and the cation exchange capacity of cell walls isolated from root-tips (0–1 em). Preliminary exposure to Al for 1 h was sufficient to inhibit subsequent root re-elongation in an Al-free solution, and the inhibitory effect was more pronounced in the Al-sensitive cultivars than in the Al-tolerant ones. Together with the inhibition of root re-elongation, the PM of the root-tip cells of all the Al-sensitive cultivars was more permeabilized than that of the Al-tolerant cultivars, based on the FDA-PI fluorescence staining technique. Exposure for 30 min to Al treatment at 100 µM significantly increased the PM permeability of protoplasts isolated from the root-tips for the Al-sensitive pea cultivar placed in a moderately hypotonic medium. Protoplasts from root-tip portions of all the Al-sensitive cultivars took up more Al than those of the Al-tolerant ones when treated with 100 pM Al under isotonic conditions for 30 min. The co-existence of DNP or hypotonic conditions led to a larger increase of Al uptake by the protoplasts from Al-sensitive maize cultivars. These results suggest that Al ions rapidly alter the PM of the root-tip portion in the Al-sensitive cultivars, irrespective of plant species, resulting in an increase of the PM permeability.

Electrophysiological and biochemical changes in rabbit hearts stored at 4 °C for 6 or 24 h

This study examines the electrophysiological and metabolic changes that occur in rabbit hearts during hypothermic storage in vitro. Hearts were microperfused at 4 degrees C for 6 or 24 h with either normal Krebs-Henseleit buffer (KHB) or KHB containing 2,3-butanedione monoxime (BDM). After hypothermic storage, hearts were rewarmed to 37 degrees C with KHB. Cardiac function was then assessed in Langendorff perfusion mode. Electrophysiological changes were also assessed from the ventricular paced-evoked responses. After storage, mitochondria were isolated from the hearts and their respiratory control ratio, rate of ATP synthesis and outer membrane intactness were assessed. Compared with values from fresh non-stored hearts, hearts stored hypothermically for 24 h showed significant decreases in both left ventricular developed pressure and coronary flow when reperfused in Langendorff mode. On the other hand, the decrease in left ventricular developed pressure in hearts that were stored for only 6 h (with or without BDM) was not significant. Compared with values obtained from fresh non-stored hearts, hypothermic storage significantly decreased the R-wave amplitude, and both the R-E and ST-E intervals of paced-evoked responses. This was true for hearts microperfused for 6 h (with or without BDM) and for hearts microperfused with buffer containing BDM for 24 h. The ST-R intervals in hearts microperfused hypothermically for 6 h were prolonged, but this change was not statistically significant compared with those obtained from unstored hearts. In hearts microperfused with KHB containing BDM for 24 h, the ST-R interval was significantly prolonged. Hypothermic microperfusion for 24 h significantly decreased both the mitochondrial coupling ratio and the rate of ATP synthesis. In hearts microperfused with BDM for 6 h, mitochondrial coupling ratios and the rate of ATP synthesis were not significantly different from those in fresh hearts. In conclusion, the present study has shown that long-term hypothermic storage significantly impaired both paced-evoked responses and mitochondrial function. Inclusion of BDM in the perfusion buffer during storage significantly ameliorated some of these changes.

Collagen membrane resorption in dogs: a comparative study.

Guided tissue barriers using materials such as collagen are used in the hope of excluding epithelium and the gingival corium from the root surface or alveolar bone to facilitate regeneration. Convention suggests that the longer a membrane remains intact, the better the regeneration results. The purpose of this study was to determine the resorption rates of various collagen membranes in the oral cavity of dogs. Twelve adult mongrel dogs had three different collagen membranes (BioGide, AlloDerm porcine-derived, and AlloDerm human-derived) randomly inserted and secured into surgical pouches made in their palates. Full-thickness tissue punch biopsy specimens taken at 1, 2, 3, or 4 months after surgery were evaluated histologically for membrane intactness and other associated changes. At 1 month, all membranes had slight to moderate degradation. At 2 months, all membranes had moderate to severe degradation with the exception of one AlloDerm human-derived membrane that was intact. At 3 months, all membranes had severe degradation to not identifiable. At 4 months, all membranes had severe degradation to completely absent. Blood vessel penetration varied from none to moderate. Inflammation was found in only two samples. In the dog, all three tested collagen membranes showed slight to moderate degradation at 1 month and were severely degraded to completely absent at 4 months. Within the limits of transferring animal data to humans, clinicians need to be aware of these resorption rates when selecting membranes for guided tissue and bone regeneration.

γ-Cyclodextrins Greatly Enhance Translocation of Hydrophobic Fluorescent Phospholipids from Vesicles to Cells in Culture: IMPORTANCE OF MOLECULAR HYDROPHOBICITY IN PHOSPHOLIPID TRAFFICKING STUDIES

Short-chain, fluorescent derivatives are commonly used to investigate intracellular phospholipid trafficking. However, their use can yield misleading results because they, unlike the native species, can rapidly distribute between organelles due to their low hydrophobicity. On the other hand, hydrophobic derivatives are very difficult to introduce to cells and thus have hardly been used. Here we show that carboxyethylated gamma-cyclodextrin (CE-gamma-CD) greatly enhances transfer of a variety of hydrophobic fluorescent phospholipid derivatives from vesicles to cultured cells. Several lines of evidence indicate that CE-gamma-CD enhances transfer of lipid molecules by increasing their effective concentration in the aqueous phase, rather than by inducing membrane fusion or hemifusion. Incubation with CE-gamma-CD and donor lipid vesicles does not extract cholesterol or phospholipids from the cells or compromise plasma membrane intactness or long term cell viability. Using CE-gamma-CD-mediated transfer, we introduced hydrophobic pyrene-labeled phosphatidylserine to the plasma membrane of fibroblast cells and followed their distribution with time. In contrast to what has been previously observed for other, less hydrophobic species, transport of this lipid to the Golgi apparatus or mitochondria was not detected. Rather, much of this fluorescent PS remained in the plasma membrane or was incorporated to various endocytotic compartments. These findings indicate that the native, typically hydrophobic phosphatidylserine molecules efflux only very slowly via the cytoplasm to intracellular organelles. This helps to explain how cells can maintain a very high concentration of phosphatidylserine in the inner leaflet of their plasma membrane. Furthermore, the present results underline the importance of using hydrophobic analogues when studying intracellular trafficking of many phospholipid classes.

NAD(P)+‐dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings

Isocitrate dehydrogenase (IDH) activities were measured in mitochondria isolated from aerial parts of 21‐day‐old spruce (Picea abies L. Karst.) seedlings. Mitochondria were purified by two methods, involving continuous and discontinuous Percoll gradients. Whatever the method of purification, the mitochondrial outer membrane was about 69% intact, and the mitochondria contained very low cytosolic, chloroplastic and peroxisomal contaminations. Nevertheless, as judged by the recovery of fumarase activity, purification on a continuous 28% Percoll gradient gave the best yield in mitochondria, which exhibited a high degree of inner membrane intactness (91%). The purified mitochondria oxidized succinate and malate with good respiratory control and ADP/O ratios. The highest oxidation rate was obtained with succinate as substrate, and malate oxidation was improved (+ 60%) by addition of exogenous NAD+. Experiments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD+‐isocitrate dehydrogenase (EC 1.1.1.41) and NADP+‐isocitrate dehydrogenase (EC 1.1.1.42) were present in the mitochondrial matrix fraction, and NAD+‐IDH activity was about 2‐fold higher than NADP+‐IDH activity. The NAD+‐IDH showed sigmoidal kinetics in response to isocitrate and standard Michaelis‐Menten kinetics for NAD+ and Mg2+. The NADP+‐IDH, in contrast, displayed lower Km values. For NAD+‐IDH the pH optimum was at 7.4, whereas NADP+‐IDH exhibited a broad pH optimum between 8.3 and 9. In addition, NAD+‐IDH was more thermolabile. Adenine nucleotides and 2‐oxoglutarate were found to inhibit NAD(P)+‐IDH activities only at high concentrations.