Phospholipid hydroperoxide glutathione peroxidase in various mouse organs during selenium deficiency and repletion

An assay for the determination of the newly discovered selenoenzyme, phospholipid hydroperoxide glutathione peroxidase (PH-GPx) in biological material is described. Dietary selenium deficiency and repletion was used as a tool in order to modify this enzyme activity in various mouse organs and to compare it to the activity of the 'classical' selenium-dependent glutathione peroxidase (GPx) (EC 1.11.1.9). A semipurified diet containing less than 12 ppb Se was used for depletion. Controls received this diet supplemented with 500 ppb Se in the form of Na2SeO3. The results showed that a rapid loss of GPx activity occurred in liver, kidney and lungs of selenium-deficient mice which reached undetectable levels within 130 days. In the heart, about 24% of control GPx activity was still present. In contrast, PH-GPx activity was more slowly depleted by Se deficiency and resulted in residual activities ranging from 30 to 70% in the different organs even after 250 days of depletion. In repletion experiments with a single application of 10 or 500 micrograms/kg Se, only the high dose restored either enzyme activity. The data demonstrate that the need for selenium of the two glutathione peroxidases is different. A markedly distinct organ distribution of both enzymes suggests that the heart may be the organ more sensitive to oxidative stress.     

Dynamics of gene expression during bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro

Characterization of directed differentiation protocols is a prerequisite for understanding embryonic stem cell behavior, as they represent an important source for cell-based regenerative therapies. Studies have investigated the osteogenic potential of human embryonic stem cells (HESCs), building upon those using pre-osteoblastic cells, however no consensus exists as to whether differentiating HESCs behave in a similar manner to the traditionally used osteoblastic progenitors. Thus, the aim of the current investigation was to define the gene expression pattern of osteoblastic differentiating HESCs, treated with ascorbic acid phosphate, β-glycerophosphate and dexamethasone over a 25 day period. Characterization of the gene expression dynamics revealed a phasic pattern of bone-associated protein synthesis. Collagen type I and osteopontin were initially expressed in proliferating immature cells, whereas osterix was up-regulated at the end of active cellular proliferation. Subsequently, mineralization-associated proteins, bone sialoprotein and osteocalcin were detected. In light of this dynamic expression pattern, we concluded that two distinguishable phases occurred during osteogenic HESC differentiation; first, cellular proliferation and secretion of a pre-maturational matrix, and second the appearance of osteoprogenitors with characteristic extracellular matrix synthesis. Establishment of this model provided the foundation of a time-frame for the additional supplementation with growth factors, BMP2 and VEGF. BMP2 induced the expression of principle osteogenic factors, such as osterix, bone sialoprotein and osteocalcin, whereas VEGF had the converse effect on the gene expression pattern.     

Disposition and hepatoprotection by phosphatidyl choline liposomes in mouse liver

Small unilamellar liposomes with an average diameter of 80 nm were prepared from phosphatidyl choline of various sources using the dialysis method with cholate as a detergent. When 14C-labeled soybean liposomes were intravenously injected into male NMRI mice, up to 10% of the total label was found in the liver lipid. The uptake was dose-dependent and reached an apparent saturation 4 h after injection. The liver maintained a constant radioactivity corresponding to 1.9 +/- 0.13 mg phospholipid/g liver until ten hours after injection of 850 mg labeled phosphatidyl choline/kg body wt. Little radioactivity was taken up by the spleen. Analogous doses of liposomes prepared from egg yolk phosphatidyl choline led to a radioactivity corresponding to 1.3 +/- 0.4 mg lipid/g liver 4 h after injection. Liposomes with a similar size were prepared from hydrated, i.e., saturated phosphatidyl choline. After intravenous administration of these liposomes, an amount of 5.3 +/- 0.5 mg labeled lipid was found per g liver after 4 h. In contrast to unsaturated liposomes, 5.8 +/- 0.8 mg lipid per gram spleen was trapped by the spleen. The pharmacodynamic effect of these different liposomes was studied in benzo[a]pyrene-pretreated mice intoxicated with 400 mg/kg paracetamol. Animals which received paracetamol exhibited serum alanine aminotransferase activities of 4220 +/- 1140 units/l after 4 h and exhaled 120 +/- 19 nmol ethane kg-1 h-1. When pretreated with 850 mg soybean phosphatidyl choline/kg body wt. (i.v.) 2 h prior to paracetamol, the increase in serum transaminase activity was reduced to 117 +/- 104 units/l and ethane exhalation amounted to 18 +/- 8 nmol kg-1 h-1. In contrast, similar pretreatment with egg yolk phosphatidyl choline or hydrated phosphatidyl choline failed to protect against paracetamol-induced hepatotoxicity. The different pharmacodynamic effects of the two phosphatidyl cholines of plant or animal origin cannot be explained on the basis of their different pharmacokinetics. In the case of soybean phosphatidyl choline liposomes, the amount of radioactive lipid found in the liver correlated with the hepatoprotective potency.     

Intracellular levels and metabolism of leucine and alpha-ketoisocaproate in normal and maple syrup urine disease fibroblasts

The interdependence of intra- and extracellular leucine and KICA concentration was studied in cultured MSUD and control fibroblasts. Intracellular KICA levels were measurable only after leucine load and were 1/20th to 1/40th that of leucine. In cells exposed to high KICA concentrations (2 mmole/liter) KICA accumulation followed linear kinetics, finally overwhelming the transamination capacity. Transamination of KICA to leucine became saturated at 2-6 mmole/liter of extracellular KICA. MSUD and control fibroblasts differed slightly in intracellular KICA concentrations, and only at high concentrations of leucine in medium. Release of KICA into the medium after leucine loading was found to be slightly increased in MSUD fibroblasts.     

MOLECULAR DIVERGENCE BETWEEN ASIAN AND NORTH AMERICAN SPECIES OF LIRIODENDRON (MAGNOLIACEAE) WITH IMPLICATIONS FOR INTERPRETATION OF FOSSIL FLORAS

Botanists have long been aware of the floristic similarities between eastern Asia and eastern North America. Most who have considered this classic disjunction pattern have suggested that it arose through range disruption of a flora that was once more widely distributed in the Northern Hemisphere. There is less agreement on the timing of this process, with suggestions ranging from the Paleocene to the Neogene. In this study, molecular markers from two different plant genomes were used to assess the degree of genetic divergence between the two interfertile, morphologically similar species of the genus Liriodendron, i.e., L. tulipifera and L. chinense. Resulting molecular divergence estimates were translated into approximate dates of separation, independent of evidence from the fossil record. Allozyme data (Nei's genetic identity = 0.434) suggested a divergence time of 10–16 million years before present, whereas sequence divergence in the plastid genomes (1.24%) led to an estimate of approximately 11–14 million years before present. A review of the paleobotanical literature indicated that the fossil floras that included, or might have included Liriodendron could not have survived in Beringia after the late Miocene and the onset of southward-migrating Arctic air masses on the North American continent. This interpretation suggests a minimum time of separation of approximately 13 million years before present. Thus, both molecular data sets and the paleobotanical evidence concur in suggesting a divergence time of 10–16 million years before present. Interspecific compatibility and relative morphological stasis must have, therefore, persisted from at least the late Miocene. We emphasize the need for similar studies in other genera, especially those that have both a reasonable Tertiary fossil history and extant species in mesic temperate refugia in Asia, Europe, and western as well as eastern North America.