Enhancement of biotinidase activity in mouse serum by inhibitors of methylation

Summary DL-ethionine increases the activity of liver biotinidase, an enzyme which hydrolyzes biotinylesters and biotinylpeptides. Chronic DL-ethionine feeding increases transiently the activity of biotinidase in mouse and rat liver, after which it remains elevated in the serum. In the present work we show that both isomers of DL-ethionine are equally good enhancers of the liver biotinidase, while, 3-ethylthiopropionate, the toxic metabolite of DL-ethionine, has no effect on the biotinidase activity of either liver or serum. We have also employed two different combinations of inhibitors of the hydrolytic pathway of SAH, a transmethylation product and potent inhibitor of methylation. It was found that these inhibitors (EHNA and Ara-A, 2-deoxycoformycin and adenosine) increase the activity of serum biotinidase as was the case with ethionine. Because SAH does not ethylate biomolecules, these changes in biotinidase activity, which can not be preveneted by adenine, biotin or lecithin are most probably related to the inhibition of methylation.Abbreviations Ara-A 9--D-arabinofuranosyladenine - EHNA erythro-9-(2-hydroxy-3-nonyl)adenine - SAE S-adenosylethionine - SAH S-adenosylhomocysteine - SAM S-adenosylmethionine     

Construction of a Stable α-Galactosidase-Producing Baker's Yeast Strain

Molasses is widely used as a substrate for commercial yeast production. The complete hydrolysis of raffinose, which is present in beet molasses, by Saccharomyces strains requires the secretion of α-galactosidase, in addition to the secretion of invertase. Raffinose is not completely utilized by commercially available yeast strains used for baking, which are Mel⁻. In this study we integrated the yeast MEL1 gene, which codes for α-galactosidase, into a commercial mel⁰ baker's yeast strain. The Mel⁺ phenotype of the new strain was stable. The MEL1 gene was expressed when the new Mel⁺ baker's yeast was grown in molasses medium under conditions similar to those used for baker's yeast production at commercial factories. The α-galactosidase produced by this novel baker's yeast strain hydrolyzed all the melibiose that normally accumulates in the growth medium. As a consequence, additional carbohydrate was available to the yeasts for growth. The new strain also produced considerably more α-galactosidase than did a wild-type Mel⁺ strain and may prove useful for commercial production of α-galactosidase.     

Changes in diatom assemblages in the profundal sediments of two large oligohumic lakes in eastern Finland

Sedimentary diatom assemblages in two large oligotrophic clear-water lakes were analysed, to assess their present ecological state and possible eutrophication due to diffuse nutrient loading. The lakes Pyhäjärvi and Puruvesi (Finnish lake district) are proportionally large for their catchment areas which accounts for their long retention times (ca 7 and 11 yr) and oligohumic character. Pyhäjärvi was studied by pairwise comparison of surface sediment diatom assemblages collected in 1985 and 1990 at 12 sites from different parts of the lake. In Puruvesi, the stratigraphy of diatoms was analysed in two short cores from 8 m and 32 m depths.The diatom assemblages of the two lakes are rather similar, and quite distinct from the assemblages of the mesohumic large lakes of the area. Cyclotella kuetzingiana is the most common planktonic dia- tom, but Aulacoseira ambigua abounds in Pyhäjärvi at sites with local sources of eutrophication. A diverse assemblage of benthic forms, especially Fragilaria and Achnanthes spp. characterizes the shallow bottoms in both lakes.There was little change within the short-core diatom profiles of Puruvesi, but the floral composition of the 8-m and 32-m sites differed markedly. The 8-m site, with 60–70% of benthic forms, represents illuminated bottom, on which much of the buried algae have lived in situ, while the deeper site is true profundal, dominated by sedimented planktonic algae.In Pyhäjärvi there was a slight increase in the benthic diatoms from 1985 to 1990, coinciding with increased phosphorus and chlorophyll concentrations as well as Secchi depth lowering. We interprete this observation as a very early step of eutrophication, of which first the sessile algal communities of the illuminated bottom areas have benefited.     

Regeneration of cleared Acacia zanzibarica bushland in Kenya

Abstract. Woody biomass production in natural forests of arid and semi-arid regions is low. The fuelwood demand of settlements often exceeds the sustained yield and regeneration capacity of natural forests, which results in deforestation. Regeneration and woody biomass development was studied in cleared Acacia zanzibarica bushland in Bura, eastern Kenya. The area was cleared in 1982 and studied in 1988. The site had been colonized primarily by Acacia zanzibarica and A. reficiens. Mean density was 1333 trees/ha, mean total woody biomass (dry weight) 1954 kg/ha, equal to 2.53 m³/ha. Mean annual increment was 293 kg/ha, or 0.3 8m³/ha. Expressed as rain use efficiency, the natural dry matter productivity of the woody component equals 0.83 kg ha^-1 yr^-1 mm^-1. The regeneration potential and some management implications are discussed.     

Longitudinal,genome-scale analysis of DNA methylation in twins from birth to 18 months of age reveals rapid epigenetic change in early life and pair-specific effects of discordance

Background

The extent to which development- and age-associated epigenetic changes are influenced by genetic, environmental and stochastic factors remains to be discovered. Twins provide an ideal model with which to investigate these influences but previous cross-sectional twin studies provide contradictory evidence of within-pair epigenetic drift over time. Longitudinal twin studies can potentially address this discrepancy.

Results

In a pilot, genome-scale study of DNA from buccal epithelium, a relatively homogeneous tissue, we show that one-third of the CpGs assayed show dynamic methylation between birth and 18 months. Although all classes of annotated genomic regions assessed show an increase in DNA methylation over time, probes located in intragenic regions, enhancers and low-density CpG promoters are significantly over-represented, while CpG islands and high-CpG density promoters are depleted among the most dynamic probes. Comparison of co-twins demonstrated that within-pair drift in DNA methylation in our cohort is specific to a subset of pairs, who show more differences at 18 months. The rest of the pairs show either minimal change in methylation discordance, or more similar, converging methylation profiles at 18 months. As with age-associated regions, sites that change in their level of within-pair discordance between birth and 18 months are enriched in genes involved in development, but the average magnitude of change is smaller than for longitudinal change.

Conclusions

Our findings suggest that DNA methylation in buccal epithelium is influenced by non-shared stochastic and environmental factors that could reflect a degree of epigenetic plasticity within an otherwise constrained developmental program.     

Subfamily III of mammalian oxysterol-binding protein (OSBP) homologues: the expression and intracellular localization of ORP3, ORP6, and ORP7

The human OSBP related protein (ORP) family consists of 12 members, which can be divided into six subfamilies based on the genomic organization and amino acid homology. Here we performed basic characterization of subfamily III, which consists of three members: ORP3, ORP6, and ORP7. According to cDNA hybridization, the three genes are expressed in a tissue-specific manner. While ORP3 mRNA is most abundant in kidney, lymph nodes, and thymus, ORP6 shows highest expression in brain and skeletal muscle, and ORP7 in the gastrointestinal tract. Using monospecific peptide antibodies, we confirmed the presence of the three proteins in human and mouse tissues. ORP6 gene expression was induced upon differentiation of F9 embryonic carcinoma cells into parietal endoderm, while ORP3 and ORP7 mRNA levels were unchanged. In the F9 cells, endogenous ORP6 associated predominantly with the nuclear envelope. When expressed from the cDNA in cultured cells, the three proteins were distributed between the cytosol and endoplasmic reticulum (ER) membranes, with a minor portion found at the plasma membrane. Experiments with truncated constructs showed that the N-terminal portion of the proteins, containing a pleckstrin homology (PH) domain, has markedly strong plasma membrane targeting specificity, while the C-terminal half remains largely cytosolic. The expression data demonstrates that ORP3, -6, and -7 are not merely redundant gene products but show marked quantitative differences in tissue expression, suggesting tissue-specific aspects in their function. The dual targeting of the proteins indicates a putative role in communication between the ER and the plasma membrane.This study was supported by the Clinical Research Fund of Helsinki University Central Hospital (J.T.), the Academy of Finland (grant 51883 to M.L.; grants 49987, 50641, and 54301 to V.M.O.), the Sigrid Juselius Foundation, and the Finnish Cultural Foundation     

Oxysterol binding protein and its homologues: new regulatory factors involved in lipid metabolism

PURPOSE OF REVIEW: Oxysterol binding protein was discovered in the 1980s as a cytosolic high-affinity receptor for oxysterols, but its function has remained enigmatic. Families of genes/proteins with sequence homology to oxysterol binding protein have been identified in eukaryotes from yeast to man, indicating that these proteins, denoted as oxysterol binding protein-related proteins (ORPs), serve a fundamental purpose conserved in evolution. This review discusses recent findings that provide important clues to the mode of action of these proteins. RECENT FINDINGS: The long variant of ORP1 is induced upon differentiation of monocytes to macrophages and has capacity to enhance the trans-activation potential of liver X receptors, indicating a function in macrophage lipid metabolism. Important clues to ORP function were provided by the finding that most family members carry an endoplasmic reticulum targeting motif, while the amino-terminal regions of the proteins have targeting specificities for other organelles. Extensive splice variation occurs within the gene family, suggesting that a large number of distinct protein products are encoded. Further implications were obtained for a possible role of a family member in tumor cell metastasis. SUMMARY: ORPs constitute a novel family of proteins implicated in cellular lipid metabolism and different aspects of cell regulation. The function of several family members is connected with cellular sterol metabolism, and there is evidence for a role of oxysterol binding protein in lipid transport from the endoplasmic reticulum. Recently, a model on the function of these proteins at membrane contact sites, specialized zones of communication between two different organelles, has been presented.     

PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma

Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. PLTP is present in plasma in two forms, one with high and the other with low phospholipid transfer activity. We have used the human hepatoma cell line, HepG2, as a model to study PLTP secreted from hepatic cells. PLTP activity was secreted by the cells into serum-free culture medium as a function of time. However, modification of a previously established ELISA assay to include a denaturing sample pretreatment with the anionic detergent sodium dodecyl sulphate was required for the detection of the secreted PLTP protein. The HepG2 PLTP could be enriched by Heparin-Sepharose affinity chromatography and eluted in size-exclusion chromatography at a position corresponding to the size of 160 kDa. PLTP coeluted with apolipoprotein E (apoE) but not with apoB-100 or apoA-I. A portion of PLTP was retained by an anti-apoE immunoaffinity column together with apoE, suggesting an interaction between these two proteins. Furthermore, antibodies against apoE but not those against apoB-100 or apoA-I were capable of inhibiting PLTP activity. These results show that the HepG2-derived PLTP resembles in several aspects the high-activity form of PLTP found in human plasma.     

Susceptibility loci for preeclampsia on chromosomes 2p25 and 9p13 in Finnish families

Preeclampsia is a common, pregnancy-specific disorder characterized by reduced placental perfusion, endothelial dysfunction, elevated blood pressure, and proteinuria. The pathogenesis of this heterogeneous disorder is incompletely understood, but it has a familial component, which suggests that one or more common alleles may act as susceptibility genes. We hypothesized that, in a founder population, the genetic background of preeclampsia might also show reduced heterogeneity, and we have performed a genomewide scan in 15 multiplex families recruited predominantly in the Kainuu province in central eastern Finland. We found two loci that exceeded the threshold for significant linkage: chromosome 2p25, near marker D2S168 (nonparametric linkage [NPL] score 3.77; P=.000761) at 21.70 cM, and 9p13, near marker D9S169 (NPL score 3.74; P=.000821) at 38.90 cM. In addition, there was a locus showing suggestive linkage at chromosome 4q32 between D4S413 and D4S3046 (NPL score 3.13; P=.003238) at 163.00 cM. In the present study the susceptibility locus on chromosome 2p25 is clearly different (21.70 cM) from the locus at 2p12 found in an Icelandic study (94.05 cM) and the locus at 2q23 (144.7 cM) found in an Australian/New Zealand study. The locus at 9p13 has been shown to be a candidate region for type 2 diabetes in two recently published genomewide scans from Finland and China. The regions on chromosomes 2p25 and 9p13 may harbor susceptibility genes for preeclampsia.