Ontogenetic changes in hepatic glutathione system (synthesis, catabolism, export) of male Uje:WIST rats.

The age-courses of concentrations of reduced (GSH) and oxidized (GSSG) glutathione, of GSH synthesizing enzyme activities, of glutathione S-transferase (GST), of GSSG-reductase (GR) and of biliary GSH and GSSG export were measured in livers from male Uje:WIST rats. Additionally, the age-courses of plasma GSH and GSSG concentrations were investigated. The hepatic level of GSH showed a biphasic pattern with a first maximum immediately after birth and a small second peak at the 50th day of life. The GSSG level increased continuously up to day 60 of life. The cytosolic GSH synthesizing enzyme activities showed diverse developmental patterns indicating different regulation principles. The hepatic activity of GR was relatively constant in the different age groups after birth. The GST activity (with o-dinitrobenzene as substrate) was relatively low at birth (about 30% of the maximum measured at day 60 of life). The maximum of GSH plasma level was found at birth. With increasing age a significant decrease in this level was observed. The excretion rate of total GSH (GSH + 2 GSSG) in bile was found to increase about 9-fold between 15 and 105 days of age. The results indicate that changes of hepatic GSH concentration with age are dependent on numerous factors. The balance between synthesis, catabolism and export is important for the maintenance of this level.     

Absence of some truncated genes in the amphidiploid Nicotiana tabacum

As an initial step towards understanding how a multigene family evolves after an interspecific hybridization and subsequent chromosomal doubling, genomic Southern blots of three related species were compared: Nicotiana tabacum (the progeny), and Nicotiana sylvestris and Nicotiana tomentosiformis (the progenitors). Genomic restriction fragments generated by two endonucleases were hybridized with a cDNA of the small subunit of ribulose bisphosphate carboxylase from N. sylvestris. The restriction pattern of the DNA of the progenitors revealed considerable polymorphism of restriction-fragment lengths. All the fragments in N. tabacum, except one, have a corresponding fragment in one of the progenitors. Some fragments present in the parents were absent from the progeny: they may correspond to truncated genes consisting of only part of the 3' portion of the gene.     

Simultaneous measurement of Ca2+ transients and of membrane depolarizations in synaptosomes.

Calcium and membrane potential sensitive dyes have been widely used to study the biochemical effects of the intracellular calcium concentration and of the membrane potential on diverse biochemical processes. However, due to the discontinuous measurement techniques applied, it was until now impossible to get an insight into the sequence and dynamics of the induced biological reactions. In order to study the relationship between the intracellular calcium concentration and the membrane potential, an apparatus was developed capable of measuring both biological processes simultaneously. Potassium chloride induced changes of the synaptosomal membrane potential and of the intracellular calcium concentration are presented.     

The regulatory role of external cations on human polymorphonuclear phagocyte chemiluminescence

The effect of the external cations Na+ and Ca2+ on polymorphonuclear chemiluminescence was investigated. Both Ca2+ in the range of 0.2-2 mM and Na+ in the range of 114-143 mM showed a dose dependent increase in polymorphonuclear chemiluminescence, irrespective of the concurrent increase in osmolality. The Na+/H+ antiport inhibitor Amiloride decreased the response significantly. These effects were observed using buffers commonly used for chemiluminescence studies and indicate the importance of defining the Ca2+ and Na+ composition of the buffers used in chemiluminescence assays.     

Control of plant organ size by KLUH/CYP78A5-dependent intercellular signaling

Plant organs grow to characteristic sizes that are genetically controlled. In animals, signaling by mobile growth factors is thought to be an effective mechanism for measuring primordium size, yet how plants gauge organ size is unclear. Here, we identify the Arabidopsis cytochrome P450 KLUH (KLU)/CYP78A5 as a stimulator of plant organ growth. While klu loss-of-function mutants form smaller organs because of a premature arrest of cell proliferation, KLU overexpression leads to larger organs with more cells. KLU promotes organ growth in a non-cell-autonomous manner, yet it does not appear to modulate the levels of known phytohormones. We therefore propose that KLU is involved in generating a mobile growth signal distinct from the classical phytohormones. The expression dynamics of KLU suggest a model of how the arrest of cell proliferation is coupled to the attainment of a certain primordium size, implying a common principle of size measurement in plants and animals.     

Genotoxicity and inactivation of catechol metabolites of the mycotoxin zearalenone

Zearalenone (ZEN) is a highly estrogenic mycotoxin produced by Fusarium species. The adverse effects of ZEN and its reductive metabolite ??-zearalenol (??-ZEL) are often compared to those of 17??-estradiol (E2) and estrone (E1). These endogenous steroidal estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (1) hormonal activity and (2) genotoxic effects after cytochrome P450-catalyzed metabolic activation to catechols. Like E1 and E2, ZEN and ??-ZEL exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites. The subsequent methylation of catechols by catechol-O-methyltransferase (COMT) is generally considered as a detoxifying pathway. Imbalances between the activation and inactivation reactions can lead to the formation of reactive semiquinones and quinones, which can alkylate DNA or produce reactive oxygen species by redox cycling. In the present study, the genotoxicity of the catechol metabolites of ZEN, ??-ZEL, E1 and E2 was determined in a cell-free system by measuring 8-oxo-2??-deoxyguanosine using a LC-DAD-MS² method. Each of the individual catechols of ZEN, ??-ZEL, E1 and E2 induced oxidative DNA damage in calf thymus DNA. The ranking order of the DNA damaging activity was 15-hydroxy-ZEN/??-ZEL ?? 2/4-hydroxy-E1/E2 > 13-hydroxy-ZEN/??-ZEL. When hepatic microsomes from different species were incubated with ZEN, the rat had the highest activity for catechol formation, followed by human, mouse, pig and steer. The amount of catechol metabolites correlated directly with the amount of oxidative damage in calf thymus DNA. The ranking order for the rate of methylation by human hepatic COMT was 2-hydroxy-E1/E2 >> 4-hydroxy-E1/E2 >> 13/15-hydroxy-ZEN/??-ZEL. Thus, the catechol metabolites of the mycoestrogen ZEN and its reductive metabolite ??-ZEL exhibit a DNA-damaging potential comparable to that of the catechol metabolites of E1 and E2, but are much poorer substrates for inactivation by human COMT.     

Monocyte-derived human macrophages mediate anergy in allogeneic T cells and induce regulatory T cells

Activation of alloreactive T cells by APCs such as dendritic cells (DC) has been implicated as crucial step in transplant rejection. In contrast, it has been proposed that macrophages (Mphi) maintain tolerance toward alloantigens. It was therefore the aim of this study to further analyze the T cell-stimulatory capacity of mature DC and Mphi in vitro using the model of allogeneic MLR. There was a strong proliferative response in T cells cocultured with DC, which was further increased upon restimulation in a secondary MLR. In contrast, T cells did not proliferate in cocultures with Mphi despite costimulation with anti-CD28 and IL-2. Cytokine analysis revealed considerable levels of IL-10 in cocultures of T cells with Mphi, whereas high amounts of IL-2 and IFN-gamma were present in cocultures with DC. There was only minimal T cell proliferation in a secondary MLR when T cells were rescued from primary MLR with Mphi and restimulated with DC of the same donor, or DC of an unrelated donor (third party), whereas a strong primary proliferative response was observed in resting T cells, demonstrating induction of T cell anergy by Mphi. Functional analysis of T cells rescued from cocultures with Mphi demonstrated that anergy was at least partly mediated by IL-10-producing regulatory T cells induced by Mphi. These results demonstrate that Mphi drive the differentiation of regulatory T cells and mediate anergy in allogeneic T cells, supporting the concept that Mphi maintain peripheral tolerance in vivo.     

Design, synthesis, and SAR studies on a series of 2-pyridinylpiperazines as potent antagonists of the melanocortin-4 receptor

A series of 2-pyridinylpiperazines derived from beta-Ala-(2,4-Cl)Phe dipeptide was synthesized for the study of their SARs and possible interactions with the MC4 receptor. Compounds such as 11k (Ki=6.5 nM) possessed high potency.     

Leaf flavonoid content in Quercus ilex L. resprouts and its seasonal variation

Here, we provide the first report on flavonoid content in holm oak (Quercus ilex L.) leaves, analyzed by HPLC–MS/MS. Flavanols and flavonols were the predominant groups, although proanthocyanidins and many soluble tannins had a relevant presence in all leaf samples. Seasonal variation of flavonoids was determined in extracts from Q. ilex leaves during resprouting after a forest fire in two Mediterranean forests. Similar seasonal trends were observed over 2 years during the two main stress seasons (winter and summer). The most abundant flavonoid was the flavanol epicatechin, which showed similar values during the two seasons. Hexosides of the flavonols, quercetin, kaempferol and rhamnetin showed considerably higher content in winter, especially at the lowest temperatures. These variations in both forests are discussed on the basis of the chlorophyll fluorescence results obtained. Anthocyanins were found practically absent in mature leaves. Nutrient or water availability differences between sites or seasons were not related to changes in leaf flavonol-hexoside content.     

Maturational alterations in gap junction expression and associated collagen synthesis in response to tendon function

Energy-storing tendons including the equine superficial digital flexor tendon (SDFT) contribute to energetic efficiency of locomotion at high-speed gaits, but consequently operate close to their physiological strain limits. Significant evidence of exercise-induced microdamage has been found in the SDFT which appears not to exhibit functional adaptation; the degenerative changes have not been repaired by the tendon fibroblasts (tenocytes), and are proposed to accumulate and predispose the tendon to rupture during normal athletic activity. The anatomically opposing common digital extensor tendon (CDET) functions only to position the digit, experiencing significantly lower levels of strain and is rarely damaged by exercise. A number of studies have indicated that tenocytes in the adult SDFT are less active in collagen synthesis and turnover than those in the immature SDFT or the CDET. Gap junction intercellular communication (GJIC) is known to be necessary for strain-induced collagen synthesis by tenocytes. We postulate therefore that expression of GJ proteins connexin 43 and 32 (Cx43; Cx32), GJIC and associated collagen expression levels are high in the SDFT and CDET of immature horses, when the SDFT in particular grows significantly in cross-sectional area, but reduce significantly during maturation in the energy-storing tendon only. The hypothesis was tested using tissue from the SDFT and CDET of foetuses, foals, and young adult Thoroughbred horses. Cellularity and the total area of both Cx43 and Cx32 plaques/mm² of tissue reduced significantly with maturation in each tendon. However, the total Cx43 plaque area per tenocyte significantly increased in the adult CDET. Evidence of recent collagen synthesis in the form of levels of neutral salt-soluble collagen, and collagen type I mRNA was significantly less in the adult compared with the immature SDFT; procollagen type I amino-propeptide (PINP) and procollagen type III amino-propeptide (PIIINP) levels per mm² of tissue and PINP expression per tenocyte also decreased with maturation in the SDFT. In the CDET PINP and PIIINP expression per tenocyte increased in the adult, and exceeded those in the adult SDFT. The level of PINP per mm² was greater in the adult CDET than in the SDFT despite the higher cellularity of the latter tendon. In the adult SDFT, levels of PIIINP were greater than those of PINP, suggesting relatively greater synthesis of a weaker form of collagen previously associated with microdamage. Tenocytes in monolayers showed differences in Cx43 and Cx32 expression compared with those in tissue, however there were age- and tendon-specific phenotypic differences, with a longer time for 50% recovery of fluorescence after photobleaching in adult SDFT cells compared with those from the CDET and immature SDFT. As cellularity reduces following growth in the SDFT, a failure of the remaining tenocytes to show a compensatory increase in GJ expression and collagen synthesis may explain why cell populations are not able to respond to exercise and to repair microdamage in some adult athletes. Enhancing GJIC in mature energy-storing tendons could provide a strategy to increase the cellular synthetic and reparative capacity.