维生素C衍生物对癌细胞浸润转移能力的抑制作用机理研究

前期研究表明Asc2P6Plm能够有效地抑制癌细胞的浸润转移，本文试图以Asc2P6Plm对人成纤维瘤细胞浸润转移作用探讨维生素C衍生物对癌细胞转移能力抑制的机理，对HT-1080细胞分别以50-300μmol/LAsc2P6Plm处理1h，随着Asc2P6Plm浓度的增大，细胞移动的数目明显减少，Asc2P6Plm对HT-1080细胞移动的抑制作用呈现出量效关系，Asc2P6Plm对ROS的清除作用，通过自旋捕集剂DMPO以电子自旋共振方法进行研究，HT-1080细胞经Asc2P6Plm处理后，细胞内的自由基水平与对照组相比有显著的降低，用F-actin的分子探针NBD研究表明，随处理时间延长，细胞内荧光强度与对照组相比显著降低，Western blots研究表明，细胞核内的RhoA蛋白量随Asc2P6Plm处理时间延长而逐渐增加，研究提示，Asc2P6Plm对癌细胞浸润转移能力的抑制作用是与抑制癌细胞内的ROS，提高细胞核内RhoA水平，降低细胞质内F-actin相关。     

Enhanced Protection Against Peroxidation-Induced Mortality of Aortic Endothelial Cells by Ascorbic Acid-2-O-Phosphate Abundantly Accumulated in the Cell as the Dephosphorylated Form

Bovine aortic endothelial BAE-2 cells exposed to the peroxidizing agent, tert-butylhydroperoxide (t-BuOOH) or 2,4-nonadienal (NDE), suffered from disruption of cell membrane integrity and from reduction of mitochondrial dehydrogenase activity as assessed by fluorometry using ethidium homodimer and photometry using WST-1, respectively. The cells were protected from t-BuOOH-induced injury more markedly by L-ascorbic acid-2-O-phosphate (Asc2P) stably masked at the 2,3-enediol moiety, which is responsible for the antioxidant ability of L-ascorbic acid (Asc), than by Asc itself. In contrast, NDE-induced membrane disruption but not mitochondrial dysfunction was prevented by Asc2P, whereas Asc exhibited no prevention against both types of injury. The amount of intracellular Asc was 7.2- to 9.0-fold larger in Asc2P-administered BAE-2 cells, where the intact form Asc2P was not detected, than in Asc-administered cells as assessed by HPLC of cell extract with detection by coulometric ECD and W. During transmembrane influx into the cell, Asc2P was concentrated as highly as 70- to 90-fold relative to the extracellular Asc2P concentration, whereas Asc was 8-to 13-fold concentrated as estimated based on an intracellular water content of 0.59 pL/cell determined by [¹⁴C]PEG/gas chromatography. Thus, Asc2P but not Asc is highly concentrated in the aqueous phase of the cell after prompt dephosphorylation, and may thereby render the cell more resistant to t-BuOOH-peroxidation assumedly via scavenging of intracellular reactive oxygen species than to peroxidation with the less hydroplulic agent NDE.     

Cytoprotection against ischemia-induced DNA cleavages and cell injuries in the rat liver by pro-vitamin C via hydrolytic conversion into ascorbate

To search a regimen for prevention of post-ischemic reperfusional (I/R) injuries, I/R in the liver was induced by 30-min clamping and subsequent unfastening of the portal vein of a rat, which underwent previous intravenous administration with ascorbic acid (Asc) of 1 mg/kg or the autooxidation-resistant pro-vitamin C, 2-O-alpha-D-glucosylated Asc (Asc2G) or 2-O-phosphorylated Asc (Asc2P) of 1 mg Asc equivalent/kg from the viewpoint of utilization of antioxidants that can promptly scavenge I/R-derived reactive oxygen species. The administration with Asc, Asc2P or Asc2G prevented some features of hepatic I/R injuries such as release of hepatic marker enzymes GOT and GPT into the blood vessel, cellular degenerative symptoms including vacuolation and cell fragmentation, and nuclear DNA strand cleavage as detected by TUNEL staining. The preventive effects on I/R injuries were in the order: Asc2G > Asc2P >> Asc. This order of preventive degrees of three anti-oxidants is partly attributable to proper efficiency of conversion to vitamin C and stability in blood stream; Asc2P was moderately converted to a free monoanion form of Asc in human serum, but, in rat serum, so efficiently converted to Asc as to undergo the resultant oxidative decomposition before reaching the liver, whereas Asc2G underwent scarce conversion to Asc in human serum but moderate conversion in rat serum, suggesting that Asc2P might be less cytoprotective against I/R injury than Asc2G in the rat liver in a way different from the human liver. In contrast Asc was so susceptible to autooxidation as to be rapidly decomposed in either rat or human serum. The concentrations of ascorbyl radicals (AscR) in serum were unchanged during I/R for sham-operated rats, but appreciably diminished time-dependently for I/R-operated rats as shown by ESR spectra. A marked increase in serum AscR occurred in rats receiving Asc, Asc2G or Asc2P, but it was time-dependently restored down to the pre-ischemic level of AscR in I/R-operated rats more rapidly than in sham-operated rats. Thus, hepatic I/R injuries were shown to be prevented more markedly by Asc2G or Asc2P than by Asc, which is attributable to efficiencies of both vitamin C conversion and subsequent AscR retention.     

Cytoprotection of vascular endotheliocytes by phosphorylated ascorbate through suppression of oxidative stress that is generated immediately after post-anoxic reoxygenation or with alkylhydroperoxides

Vascular endotheliocytes BAE-2 underwent the gradually proceeding cell death until 48 h after reoxygenation (Reox) following 3 h anoxia (Anox), but protected by pre-Anox administration with L-ascorbic acid (Asc)-2-O-phosphate (Asc2P), an autooxidation-resistant Asc derivative, but not by Asc itself. This cytoprotection with Asc2P was achieved in a glucose (Glc)-lacking buffer more advantageously than in a Glc-containing buffer where less efficiency had been demonstrated for Asc entry into BAE-2 cells than in a Glc-lacking buffer. Superoxide anion radicals were detected explosively in the extracellular space at 2-5 min after Reox following the Anox treatment of HUVE endotheliocytes, and were thereafter retained at levels as high as approximately one-half of the maximum level until 60 min after Reox, as shown by cytochrome c reduction assay. Superoxide anions at 3 and 60 min after Reox were suppressed by pre-Anox administration with Asc2P, but not with Asc or dehydro-Asc, and were not suppressed by post-Anox administration with Asc2P; the cytoprotection may need the intracellular accumulation of the ROS-scavenging effector Asc that is converted from Asc2P until 3 min after Reox. The ROS-generator tert-butylhydroperoxide (t-BuOOH) also induced both the diminished cell viability and nuclear DNA strand cleavages of BAE-2 endotheliocytes, which were also protected dose-dependently with Asc2P. The cytoprotection was attributed to reduction of intracellular ROS including hydroperoxide and hydrogen peroxide with Asc2P as shown by fluorometry with the redox indicator CDCFH-DA. Thus Anox/Reox-induced cell death can be prevented by Asc2P that suppresses ROS-generation immediately after Reox following Anox more efficiently in the intracellular sphere rather than in the extracellular space.     

Role of MAPK phosphorylation in cytoprotection by pro-vitamin C against oxidative stress-induced injuries in cultured cardiomyoblasts and perfused rat heart

The reactive oxygen species (ROS) are known to be generated upon post-ischemic reperfusion (I/R) of the heart, and to injure cardiac muscle cells. The hydrogen peroxide-induced mortality of rat cardiomyoblasts H2c9 was markedly inhibited by previous administration with auto-oxidation-resistant pro-vitamin C, the 2-O-phosphorylated derivative (Asc2P) of ascorbic acid (Asc). The cytoprotection was partially counteracted by an inhibitor of MAPK (mitogen-activated protein kinase) kinase (MEK) as shown by DNA strand cleavage assay and mitochondrial dehydrogenase assay. Immunostains indicated that phosphorylated MAPK increased in the hydrogen peroxide-treated cardiomyoblasts, and that this action was moderately inhibited by Asc2P and restored nearly to the initial, pretreatment level by combined administration of the MEK inhibitor and Asc2P. The I/R-induced cell injuries in perfused rat hearts as estimated by extracellular release of the cardiac enzyme CPK were inhibited by 2-O-alpha-glucosylascorbic acid (Asc2G) and Asc, whereas the observed cytoprotection for the cardiomyoblasts was partially counteracted by the MEK inhibitor. The increase in phosphorylated MAPK in I/R-operated hearts was moderately inhibited by pro-vitamin C, but restored nearly to the normal non-operated level by combined administration with the MEK inhibitor. This is in contrast to no alteration in levels of non-phosphorylated MAPK for all the cases examined as shown by Western blots, consistent with results of immunostains for the cardiomyoblasts. The inhibitory effect of the MEK inhibitor on MAPK phosphorylation was, therefore, suggested to counteract the cytoprotective effects of pro-vitamin C via a thorough interruption of the phosphorylated MAPK signaling pathway. This was not true of ROS-related events; the scavenging effects of Asc2G and Asc on hydroxyl radicals generated from I/R-operated heart were not affected by combined administration with the MEK inhibitor, as shown by the spin-trapping DMPO-based ESR method.     

Preventive effects of phosphorylated ascorbate on ultraviolet-B induced apoptotic cell death and DNA strand cleavage through enrichment of intracellular vitamin C in skin epidermal keratinocytes

Mortality of mouse keratinocytes Pam212 that were irradiated with ultraviolet-B (UVB) was shown to be repressed by pre-irradiated administration with L-ascorbic acid (Asc) or more markedly with Asc-2-O-phosphate (Asc2P), but not with dehydroascorbic acid (DehAsc) or Asc-2-O-alpha-glucoside (Asc2G), although not repressed by post-irradiated administration. The cytoprotection by Asc2P was restricted against UVB below 5-20 mJ/cm2, and exhibited markedly by administration for a period over 2 h, which may be caused by intracellular Asc that was accumulated via dephosphorylation of Asc2P and was increased, 6-24 h after, to levels above twice as abundant as those of Asc-administration. Pre-irradiated Asc2P-administration slightly repressed a DNA ladder-like electrophoretic pattern for UVB-irradiated keratinocytes, containing the histone-bound DNA fragments as shown by ELISA assay, and appreciably repressed the DNA-3'OH cleavage terminals as shown by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stain. Thus, prevention of UVB-induced cell death by Asc2P was shown to occur concurrently with inhibition of DNA cleavages and enrichment of intracellular Asc.     

Slow-down of age-dependent telomere shortening is executed in human skin keratinocytes by hormesis-like-effects of trace hydrogen peroxide or by anti-oxidative effects of pro-vitamin C in common concurrently with reduction of intracellular oxidative stress

The cellular life-span of cultivated human skin epidermis keratinocytes NHEK-F was shown to be extended up to 150% of population doubling levels (PDLs) by repetitive addition with two autooxidation-resistant derivatives of ascorbic acid (Asc), Asc-2-O-phosphate (Asc2P), and Asc-2-O-alpha-glucoside (Asc2G), respectively, but to be not extended with Asc itself. In contrast, hydrogen peroxide (H(2)O(2)) as dilute as 20 microM which was non-cytotoxic to the keratinocytes, or at 60 microM being marginally cytotoxic achieved the cellular longevity, unexpectedly, up to 160 and 120% of PDLs, respectively, being regarded as a hormesis-like stimulatory effect. The lifespan-extended cells that were administered with Asc2P, Asc2G, or 20 microM H(2)O(2) were prevented from senescence-induced symptoms such as PDL-dependent enlargement of a cell size of 14.7 microm finally up to 17.4 microm upon Hayflick's limit-called loss of proliferation ability as estimated with a channelizer, and retained young cell morphological aspects such as thick and compact shape and intense attachment to the culture substratum even upon advanced PDLs, whereas other non-extended cells looked like thin or fibrous shape and large size upon lower PDLs. The PDL-dependent shortening of telomeric DNA of 11.5 kb finally down to 9.12-8.10 kb upon Hayflick's limit was observed in common for each additive-given cells, but was decelerated in the following order: 20 microM H(2)O(2) > Asc2P = Asc2G > 60 microM H(2)O(2) > Asc = no additive, being in accord with the order of cell longevity. Intracellular reactive oxygen species (ROS) was diminished by Asc2P, Asc2G or 20 microM H(2)O(2), but not significantly by Asc or 60 microM H(2)O(2) as estimated by fluorometry using the redox indicator dye CDCFH. There was no appreciable difference among NHEK keratinocytes that were administered with or without diverse additives in terms of telomerase activity per cell, which was 1.40 x 10(4)-4.48 x 10(4) times lower for the keratinocytes than for HeLa cells which were examined as the typical tumor cells. Thus longevity of the keratinocytes was suggested to be achieved by slowdown of age-dependent shortening of telomeric DNA rather than by telomerase; telomeres may suffer from less DNA lesions due to the continuous and thorough repression of intracellular ROS, which was realized either by pro-vitamin C such as Asc2P or Asc2G that exerted an antioxidant ability more persistent than Asc itself or by 20 microM H(2)O(2) which diminished intracellular ROS assumedly through a hormesis-like effect.     

Cytoprotection by bcl-2 gene transfer against ischemic liver injuries together with repressed lipid peroxidation and increased ascorbic acid in livers and serum

The maximum gene exhibition was shown to be achieved at 48 h after transfection with human bcl-2 (hbcl-2) genes built in an SV40 early promoter-based plasmid vector and HVJ-liposome for cultured rat hepatocytes. The similar procedure of hbcl-2 transfection was therefore conducted for livers in rats via the portal vein, and after 48 h followed by post-ischemic reperfusion (I/R) operation for some hepatic lobes. The I/R-induced hepatic injuries were in situ observed as both cell morphological degeneration and cellular DNA strand cleavages around capillary vessels of the ischemic liver lobes as detected by HE stain and TUNEL assay, and were biochemically observed as release of two hepatic marker enzymes AST and ALT into serum. All the I/R-induced injuries examined were appreciably repressed for rats transfected with hbcl-2; hbcl-2 was expressed in hepatocytes around the capillaries of ischemic regions such as the median lobe and the left lobe, but scarcely around those of non-ischemic regions. Thus cytoprotection against I/R-induced injuries may be attributed to the I/R-promoted expression of transferred hbcl-2 genes. The possibility was examined firstly by methylphenylindole method, which showed that I/R-enhanced lipid peroxidation in the reference vector-transfected livers were markedly repressed in the hbcl-2-transfected livers. Contents of ascorbic acid (Asc) in serum and livers of hbcl-2-transfected rats were enriched, unexpectedly, versus those of non-transfected rats, and were as abundant as 1.90-fold and 1.95- to 2.60-fold versus those in the pre-ischemic state, respectively. After I/R, an immediate decline in serum Asc occurred in hbcl-2-transfectants, and was followed by prompt restoration up to the pre-ischemic Asc levels in contrast to the unaltered lower Asc levels in non-transfectants except a transient delayed increase. Hepatic Asc contents were also diminished appreciably at the initial stage after I/R in the ischemic lobes of hbcl-2-transfectants, which however retained more abundant Asc versus non-transfectants especially at the initial I/R stage when scavenging of the oxidative stress should be most necessary for cytoprotection. The results showed a close correlation between cytoprotection by exogenously transferred hbcl-2 and repressive effects on the lipid peroxidation associated with Asc consumption or redistribution.     

The protective effect of ascorbic acid derivative on PC12 cells: involvement of its ROS scavenging ability

L-ascorbic acid 2-phosphate-6-palmitate (Asc2P6P) was synthesized and its effect on the damage of PC12 cells induced by H2O2 was investigated. 200 microM H2O2 in a treatment period of 4 hours in our experiment resulted in substantial cell loss. With the increasing concentration of antioxidants, such H2O2-induced cytotoxicity was significantly prevented and the corresponding intracellular and extracellular ROS levels decreased concurrently by pre-treatment with Asc2P6P and Asc. It was found that Asc2P6P was superior to L-ascorbic acid in its protective role and showed a dose-dependent manner during a 24-hour treatment. The higher potency of Asc2P6P's protective role on PC12 cells was correlated with its more effective ROS scavenging ability. HPLC assay demonstrated that Asc2P6P could easily enter the cells and be converted into Asc persistently, which contributed to its distinguished role in protecting PC12 cells against H2O2-induced cytotoxicity.     

Potentiated susceptibility of ascites tumor to acyl derivatives of ascorbate caused by balanced hydrophobicity in the molecule

Orders of susceptibility of Ehrlich ascites tumor to L-ascorbic acid (Asc), its 6-stearoyl (6S), 6-palmitoyl (6P) and 2,6-dipalmitoyl (DP) derivatives were assessed in vitro and in vivo: 6P (a 50% growth inhibitory concentration (IC50) for cultured cells, 12 microM; an increased life-span of treated mice, 283%) greater than 6S (61 microM; 240%) much greater than Asc (430 microM; 122%) greater than or equal to DP (greater than 200 microM; 89%), indicating that the enhanced susceptibility was due to acyl moiety substituted at C6-hydroxyl group of Asc, but was retracted by further substitution at C2-hydroxyl group. Equimolar mixture of Asc and palmitic acid, stearic acid or their methyl esters was much less cytotoxic than 6P or 6S. Thus the enhanced susceptibility was not primarily due to an additive cytotoxic effect of ascorbyl and acyl moieties, but to a balanced hydrophobicity introduced into the molecule by a poorly cytotoxic acyl moiety.     

DNA repair in cultured mouse cells of increasing population doubling level

Cultures of mouse cells of various population doubling levels (PDL) were examined for DNA-repair capabilities as estimated by (i) the excision of pyrimidine dimers; (ii) unscheduled DNA synthesis (UDS) in response to UV-irradiation or N-methyl-N'-nitrosoguanidine (MNNG) treatment; (iii) the levels of two DNA-repair enzyme activities, uracil DNA glycosylase and AP endonuclease. The responses to ultraviolet light and MNNG decreased rapidly within the first two PDL and more slowly thereafter until essentially no repair was detected by PDL 12. A continuous cell line which emerged from the cultured cells after a crises period had some restoration of repair capability. The amount of uracil DNA glycosylase activity decreased by approximately 40% before the crises period then decreased by 90% in the continuous cell line. In contrast, the amount of AP endonuclease activity present in the precrises cells showed no significant change until PDL 12, then increased 6-7-fold in the continuous cell line.     

L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts

Proliferation of human skin fibroblasts was stimulated significantly by the presence of L-ascorbic acid 2-phosphate (Asc 2-P). The presence of Asc 2-P (0.1-1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2-fold as well as cell growth 4-fold. Coexistence of L-azetidine 2-carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2-P in a dose-dependent manner. Supplementation of the medium with Asc 2-P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum-rich cells surrounded by dense ECM. These results indicate that Asc 2-P is useful in culture systems as a long-acting vitamin C derivative and also that it promotes reorganization of a three-dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.     

Oral vitamin C enhances the adrenergic vasoconstrictor response to local cooling in human skin

Local administration of ascorbic acid (Asc) at a supraphysiological concentration inhibits the cutaneous vasoconstrictor response to local cooling (LC). However, whether orally ingesting Asc inhibits the LC-induced vasoconstrictor response remains unknown. The purpose of the present study was to examine the acute influence of oral Asc on the adrenergic vasoconstrictor response to LC in human skin. In experiment 1, skin blood flow (SkBF) was measured by laser-Doppler flowmetry at three sites (forearm, calf, palm). The three skin sites were locally cooled from 34 to 24°C at -1°C/min and maintained at 24°C for 20 min before (Pre) and 1.5 h after (Post) oral Asc (2-g single dose) or placebo supplementation. Cutaneous vascular conductance (CVC) was calculated as the ratio of SkBF to blood pressure and expressed relative to the baseline value before LC. Oral Asc enhanced (P < 0.05) the reductions in CVC in the forearm (Pre, -50.3 ± 3.3%; Post, -57.8 ± 2.2%), calf (Pre, -52.6 ± 3.7%; Post, -66.1 ± 4.3%), and palm (Pre, -46.2 ± 6.2%; Post, -60.4 ± 5.6%) during LC. The placebo did not change the responses at any site. In experiment 2, to examine whether the increased vasoconstrictor response caused by oral Asc is due to the adrenergic system, the release of neurotransmitters from adrenergic nerves in forearm skin was blocked locally by iontophoresis of bretylium tosylate (BT). Oral Asc enhanced (P < 0.05) the reductions in CVC at untreated control sites but did not change the responses at BT-treated sites during LC. In experiment 3, to further examine whether adrenergically mediated vasoconstriction is enhanced by oral Asc, 0.1 mM tyramine was administered using intradermal microdialysis in the forearm skin at 34°C in the Pre and Post periods. Oral Asc increased (P < 0.05) the tyramine-induced reduction in CVC. These findings suggest that oral Asc acutely enhances the cutaneous vasoconstrictor responses to LC through the modification of adrenergic sympathetic mechanisms.     

The culture of chick embryo dorsal root ganglionic cells on polylysine-coated plastic

Polylysine-coated culture surfaces are strongly adhesive for neural cells, restrict locomotion on nonneuronal elements, but do not inhibit neurite elongation. In the present study, culture dishes were pre-treated with poly-d-lysine (PDL) at various concentrations, seeded with dissociates from 8-day chick embryo dorsal root ganglia, and incubated under conditions that normally support both neuronal survival and nonneuronal proliferation. Pretreatment with low (0.1 mg/ml) PDL concentrations had no effect on neuronal survival and neuritic growth, but entirely prevented an increase in ganglionic nonneurons, yielding a numerically stable culture greatly enriched in neurons. Higher PDL concentrations caused increasing losses in both cell classes. The 50% levels of cell loss were achieved at about the same PDL dose, but earlier for neurons than nonneurons and still with no impairment of neuritic growth from the surviving neurons. A procedure was developed to compare acid-soluble and acid-precipitable accumulation of radioactivity under 1-hr pulses of [³H]uridine, which was applicable even to poorly attached cells. The cytotoxic effects of higher PDL pretreatments was revealed as early as 6 hr after seeding by 2- to 4-fold lower radioaccumulation. The data are discussed in terms of possible regulations of cell permeability and metabolism by adhesive interactions between cells and their substratum, or other cells.     

Refolding of soluble leukemia inhibitory factor receptor fusion protein (gp 190 sol DAF) from urea

Uptake of L-[1-14C]ascorbic acid (Asc) of 12.5-200 µM for 1 h intobovine aortic endothelial BAE-2 cells grown to confluence was as low as43-64% (per cell) of uptake into the cells grown to nearly one-fourthconfluence. [14C]Asc undergoing transmembrane uptake was concentrated andaccumulated in the cell less efficiently ([Asc]in/ex = 8-13) at confluencethan at subconfluence ([Asc]in/ex = 15-24). The declined Asc uptake atconfluence is attributable to slowdown of the cell cycle, because a similardecrease in [Asc]in/ex was shown by subconfluent cells precultured inserum-insufficient medium, resulting in an increase in G1 phase andconcurrent decreases in S and G2 + M phase distributions as determined byflow cytometry. [1-14C]Dehydroascorbic acid (DehAsc) was taken up andaccumulated as Asc, after metabolic reduction, without detectable DehAsc.The [Asc]in/ex values for DehAsc at confluence were as low as 15-69%of those at subconfluence in contrast to the values as retentive as62-75% for Asc, suggesting the moderate control of Asc uptake againstslowdown of the cell cycle. At either confluence or subconfluence,dose-dependence for DehAsc uptake was more marked than for Asc uptake asshown by an uphill slope in a curve of doses versus [Asc]in/ex for DehAsc incontrast to a downhill slope for Asc, suggesting the moderate control forAsc uptake against fluctuation of the dose. Increasing of coexistent glucoseof 5 mM to 20-40 mM, plasma concentrations in diabetic patients, declinedDehAsc uptake to 46-48%, which was less moderately controlled thanAsc uptake retained to 59-73%. Asc uptake did not compete with DehAscuptake, suggesting different transporter proteins for Asc and DehAsc. Thus,Asc uptake into the aortic endothelial cells is more moderately controlledagainst slowdown of the cell cycle, decreasing of the extracellularconcentrations or increasing of coexistent glucose than DehAsc uptake,suggesting a homeostatic advantage of Asc over DehAsc in terms of retentionof intracellular Asc contents within a definite range.     

Changes in Leydig cell function during sexual maturation in the mouse

Changes in androgen production by isolated Leydig cells were evaluated from 20 through 60 days of age in the mouse. Leydig cells were obtained by mechanical dissociation of testes, purified by centrifugation in metrizamide gradients, and incubated with increasing concentrations of human chorionic gonadotropin (hCG). Testosterone and 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol) were measured by radioimmunoassay in samples of cells plus medium. Sensitivity of mouse Leydig cells, evaluated as the concentration of hCG that elicited half-maximum androgen responses, was essentially the same at all ages. Maximum testosterone production increased by about 20-fold from 20 to 45 days of age but was no greater at 60 days than at 45 days. Maximum androstanediol production increased by about 3- to 4-fold from 20 to 25 days and declined after 30 days of age. Androstanediol predominated over testosterone by about 2-fold at 20 days; this relationship was reversed by 30 days, and at later ages testosterone greatly predominated over androstanediol (by at least 4- and 6-fold at 45 and 60 days of age, respectively). Maximum total androgen production, estimated from the sum of the values for testosterone and androstanediol, increased by about 7-fold from 20 to 30 days of age and remained essentially constant thereafter. These results are compared with those from previous studies of the rat.     

Light-induced ascorbate-dependent electron transport and membrane energization in chloroplasts of bundle sheath cells of the C4 plant maize

Chloroplasts in bundle sheath cells (BSC) of maize perform photosystem I (PSI)-mediated production of ATP. In this study, the participation of ascorbate (Asc) as an electron donor to PSI in light-induced electron transport in isolated maize BSC was demonstrated. It was found that Asc, at physiological concentrations, rapidly reduced photooxidized reaction center chlorophyll of PSI (P700). The rate of Asc donation of electrons to P700+ reached rates of 50-100 microequivalents (mg Chl)(-1) h(-1) at 70-80 mM ascorbate with methyl viologen as an electron acceptor. Electron transport supported by Asc was coupled with membrane energization, as demonstrated by the light-induced formation of a trans-thylakoid electric field measured by the electrochromic shift of carotenoids. The possible physiological function of Asc-dependent electron transport in bundle sheath chloroplasts of maize, as an electron donor for linear electron flow versus sustaining cyclic electron transport, is discussed.     

Acceleration of Enterococcus faecalis biofilm formation by aggregation substance expression in an ex vivo model of cardiac valve colonization

Infectious endocarditis involves formation of a microbial biofilm in vivo. Enterococcus faecalis Aggregation Substance (Asc10) protein enhances the severity of experimental endocarditis, where it has been implicated in formation of large vegetations and in microbial persistence during infection. In the current study, we developed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10(+) and Asc10(-)E. faecalis and valve tissue, and to examine formation of E. faecalis biofilms on a relevant tissue surface. Scanning electron microscopy of the infected valve tissue provided evidence for biofilm formation, including growing masses of bacterial cells and the increasing presence of exopolymeric matrix over time; accumulation of adherent biofilm populations on the cardiac valve surfaces during the first 2-4 h of incubation was over 10-fold higher than was observed on abiotic membranes incubated in the same culture medium. Asc10 expression accelerated biofilm formation via aggregation between E. faecalis cells; the results also suggested that in vivo adherence to host tissue and biofilm development by E. faecalis can proceed by Asc10-dependent or Asc10-independent pathways. Mutations in either of two Asc10 subdomains previously implicated in endocarditis virulence reduced levels of adherent bacterial populations in the ex vivo system. Interference with the molecular interactions involved in adherence and initiation of biofilm development in vivo with specific inhibitory compounds could lead to more effective treatment of infectious endocarditis.