Altered expression of diacylglycerol kinase isozymes in regenerating liver

The liver possesses the capacity to restore its function and mass after injury. Liver regeneration is controlled through complicated mechanisms, in which the phosphoinositide (PI) cycle is shown to be activated in hepatocytes. Using a rat partial hepatectomy (PH) model, the authors investigated the expression of the diacylglycerol kinase (DGK) family, a key enzyme in the PI cycle, which metabolizes a lipid second-messenger diacylglycerol (DG). RT-PCR analysis shows that DGKζ and DGKα are the major isozymes in the liver. Results showed that in the process of regeneration, the DGKζ protein, which is detected in the nucleus of a small population of hepatocytes in normal liver, is significantly increased in almost all hepatocytes. However, the mRNA levels remain largely unchanged. Double labeling with bromodeoxyuridine (BrdU), an S phase marker, reveals that DGKζ is expressed independently of DNA synthesis or cell proliferation. However, DGKα protein localizes to the cytoplasm in normal and regenerating livers, but immunoblot analysis reveals that the expected (80 kDa) and the lower (70 kDa) bands are detected in normal liver, whereas at day 10 after PH, the expected band is solely recognized, showing a different processing pattern of DGKα in liver regeneration. These results suggest that DGKζ and DGKα are involved, respectively, in the nucleus and the cytoplasm of hepatocytes in regenerating liver.     

Charged-particle mutagenesis. 1. Cytotoxic and mutagenic effects of high-LET charged iron particles on human skin fibroblasts.

Cytotoxic and mutagenic effects of high-LET charged iron (56Fe) particles were measured quantitatively using primary cultures of human skin fibroblasts. Argon and lanthanum particles and gamma rays were used in comparative studies. The span of LETs selected was from 150 keV/microns (330 MeV/u) to 920 keV/microns (600 MeV/u). Mutations were scored at the hypoxanthine guanine phosphoribosyl transferase (HPRT) locus using 6-thio-guanine (6-TG) for selection. Exposure to these high-LET charged particles resulted in exponential survival curves. Mutation induction, however, was fitted by the linear model. The relative biological effectiveness (RBE) for cell killing ranged from 3.7 to 1.3, while that for mutation induction ranged from 5.7 to 0.5. Both the RBE for cell killing and the RBE for mutagenesis decreased with increasing LET over the range of 1.50 to 920 keV/microns. The inactivation cross section (sigma i) and the action cross section for mutation induction (sigma m) ranged from 32.9 to 92.0 microns2 and 1.45 to 5.56 X 10(-3) microns2; the maximum values were obtained by 56Fe with an LET of 200 keV/microns. The mutagenicity (sigma m/sigma i) ranged from 2.05 to 7.99 X 10(-5) with an inverse relationship to LET.     

Nonspecific lipid transfer protein in castor bean cotyledon cells: subcellular localization and a possible role in lipid metabolism.

The subcellular localization and several biochemical activities of nonspecific lipid transfer protein (nsLTP) were investigated. A section of a castor bean cotyledon cell was labeled with anti-nsLTP serum followed by protein A-gold. Gold particles were more abundant in the glyoxysome matrix and the vessel cell wall than in other areas. Cell fractionation analysis of 6-day-old castor bean cotyledons by sucrose density gradient centrifugation demonstrated that 13% of nsLTP was distributed in the glyoxysomal fraction, identified on the basis of catalase as a marker, and 87% in the soluble fraction near the top of the gradient. The location of castor bean nsLTP in glyoxysomes was further confirmed by in vitro import experiments. The synthesized precursor of nsLTP (pro-nsLTP-C) was incorporated into intact castor bean glyoxysomes and processed to the mature form after import into the glyoxysomes, but it was not imported into canine pancreatic microsomes. Castor bean nsLTP-A was found to possess the ability to bind oleic acid and oleoyl-CoA by means of a method involving Lipidex 1000. The dissociation constants (Kd) for oleic acid and oleoyl-CoA binding to nsLTP-A were 4.8 and 5.0 microM, respectively. The saturated binding capacities (Bmax) for oleic acid and oleoyl-CoA per mol of nsLTP-A were 1.1 and 1.2 mol, respectively. When acyl-CoA oxidase activity was assayed in the glyoxysomal fraction, marked enhancement of the activity was observed in the presence of nsLTP. These results suggest the possibility that nsLTP regulates fatty acid beta-oxidation through the enhancement of acyl-CoA oxidase activity in glyoxysomes. The occurrence of castor bean nsLTP in the vessel wall was discussed.     

S-(1,2-dicarboxyethyl)glutathione and activity for its synthesis in rat tissues

The contents of S-(1,2-dicarboxyethyl)glutathione (DCE-GS) in several tissues of rat were determined by HPLC. The peptide was present at concentrations (nmol/g tissue) of 119 in lens, 71.6 in liver, and 27.4 in heart. It was, however, not detected in spleen, kidney, cerebrum, or cerebellum. In rat liver, DCE-GS was located primarily in the cytosolic fraction. The substrates for the enzymic synthesis of DCE-GS were GSH and L-malate. In rats, the DCE-GS-synthesizing activity was found to be highest in the liver and in the cytosol of rat liver subcellular fractions. The DCE-GS-synthesizing enzyme was partially purified from rat liver cytosolic fraction by ammonium sulfate fractionation, Phenyl Superose chromatography, hydroxyapatite chromatography, and gel filtration. The molecular mass of the enzyme was estimated to be 53 kDa by gel filtration and SDS-PAGE, showing it to be a monomeric protein. The Km values for GSH and L-malate were 2.3 and 4.0 mM at 37 degrees C, respectively. The enzyme did not utilize 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, p-nitrophenyl bromide, trans-4-phenyl-3-buten-2-one, or p-nitrobenzyl chloride, which were substrates for previously characterized glutathione S-transferases. The isolated enzyme preparation showed no fumarase activity, which supported the conclusion that the formation of DCE-GS was not the result of a nonenzymic reaction following the synthesis of fumarate from L-malate by the isolated enzyme. The N-terminal amino acid of this polypeptide was presumably blocked since no sequence was obtained by automatic sequencing after electro-blotting onto a siliconized-glass fiber (SGF) sheet.     

Studies on Production of Biotin by Microorganisms

In a preceding paper we reported that Rhodotorula flava 194 effectively converted biotin to biotinamide. In a present paper the metabolism of desthiobiotin by R. flava 194 was studied under the same condition as in the conversion of biotin to biotinamide. Two desthiobiotin derivatives (Vitamer I and II) were isolated. Vitamer II (crystalline) was identified as bisnordesthiobiotin and Vitamer I was chromatographically determined as desthiobiotinamide.     

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb^−/−). Rheb^−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb^−/− was lower than that in the control (Rheb^+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb^+/+ mice but not in Rheb^−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb^−/− hearts during the neonatal period. Rheb^−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb^−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb^−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.     

Short-term ascorbic acid deficiency induced oxidative stress in the retinas of young guinea pigs

We examined whether short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs. Four-week-old guinea pigs were given a scorbutic diet (20 g/animal/day) with and without adequate ascorbic acid (400 mg/animal/day) in drinking water for 3 weeks. The serum concentrations of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 14.1 and 4.1%, respectively, of those in the adequate group. The retinal contents of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 6.4 and 27.3%, respectively, of those in the adequate group. The retinal content of thiobarbituric acid-reactive substances, an index of lipid peroxidation, was 1.9-fold higher in the deficient group than in the adequate group. Retinal reduced glutathione and vitamin E contents in the deficient group were 70.1 and 69.4%, respectively, of those in the adequate group. This ascorbic acid deficiency did not affect serum thiobarbituric acid-reactive substances and reduced glutathione concentrations but increased serum vitamin E concentration. These results indicate that short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs without disrupting systemic antioxidant status.     

Global patterns of genetic variation in plant species along vertical and horizontal gradients on mountains

Aim To understand global patterns of genetic variation in plant species on mountains and to consider the significance of mountains for the genetic structure and evolution of plant species. Location Global. Methods We review published studies. Results Genetic diversity within populations can vary along altitudinal gradients in one of four patterns. Eleven of 42 cited studies (26% of the total) found that populations at intermediate altitudes have greater diversity than populations at lower and higher altitudes. This is because the geographically central populations are under optimal environmental conditions, whereas the peripheral populations are in suboptimal situations. The second pattern, indicating that higher populations have less diversity than lower populations, was found in eight studies (19%). The third pattern, indicating that lower populations have lower diversity than higher populations, was found in 10 studies (24%). In 12 studies (29%), the intrapopulation genetic variation was found to be unaffected by altitude. Evidence of altitudinal differentiation was found in more than half of these studies, based on measurements of a range of variables including genome size, number of chromosomes or a range of loci using molecular markers. Furthermore, great variation has been found in phenotypes among populations at different altitudes in situ and in common garden experiments, even in cases where there was no associated variation in molecular composition. Mountains can be genetic barriers for species that are distributed at low elevations, but they can also provide pathways for species that occupy high‐elevation habitats. [Correction added after publication 9 October 2007: ‘less diversity’ changed to ‘greater diversity’ in the second sentence of the Results section of the Abstract] Main conclusions Genetic diversity within populations can vary along altitudinal gradients as a result of several factors. The results highlight the importance of phenotypic examinations in detecting altitudinal differences. The influence of mountain ridges on genetic differentiation varies depending, inter alia, on the elevation at which the species occurs. Based on these findings, zoning by altitudes or ridges would be helpful for the conservation of tree populations with the onset of global warming.     

Pyridoxine stimulates filaggrin production in human epidermal keratinocytes

Molecular Biology Reports - Pyridoxine (PN), one of the vitamers of vitamin B6, plays an important role in the maintenance of epidermal function and is used to treat acne and rough skin. Clinical...     

Characterization of the cysteine-rich calcium-binding S100A3 protein from human hair cuticles

S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.