Affinity-chromatographic purification of S-adenosyl-L-homocysteine hydrolase. Some properties of the enzyme from rat liver.

S-Adenosyl-L-homocysteine hydrolase has been purified to apparent homogeneity from rat liver by means of affinity chromatography on 8-(3-aminopropylamino)adenosine linked to Sepharose. The purified enzyme was free from adenosine kinase and adenosine deaminase activities and was homogeneous on SDS/polyacrylamide-gel electrophoresis which gave a subunit mol.wt. of 47 000. The native enzyme showed some microheterogeneity on polyacrylamide-gel electrophoresis under increased-resolution conditions but was homogeneous on isoelectric focusing (pI 5.6). The molecular weight of the native enzyme was about 220 000 as judged by pore-gradient electrophoresis. The native enzyme bound adenosine tightly and showed Km values of 0.6 microM, 0.9 microM and 60 microM for adenosine, S-adenosyl-L-homocysteine and L-homocysteine respectively. The enzyme was rapidly inactivated when incubated in the presence of adenosine, S-adenosyl-L-homocysteine or several adenosine derivatives or analogues. Inactivation took place both at 0 and 37 degrees C. Freezing in the absence of glycerol resulted in the appearance of dissociation products of the oligomeric protein. Multimer formation was observed at low thiol concentrations.     

Metabolism,cellular actions,and cytotoxicity of selenomethionine in cultured cells

Selenomethionine metabolism and the biochemical basis for its cytotoxicity were analyzed in cultured human and murine lymphoid cells. The metabolic pathways were also addressed, using purified mammalian enzymes and crude tissue extracts. Selenomethionine was found to be effectively metabolized to S-adenosylmethionine analog, and that analog was further metabolized in transmethylation reactions and in polyamine synthesis, similarly to the corresponding sulphur metabolites of methionine. Selenomethionine did not block these pathways, nor was there a specific block on the synthesis of DNA, RNA, or proteins when added to the culture medium. Selenomethionine showed cytotoxicity at above 40 microM levels. Yet, low selenomethionine levels (10 microM) could replace methionine and support cell growth in the absence of methionine. Selenomethionine toxicity took place concomitantly with changes in S-adenosylmethionine pools. D-form was less cytotoxic than L-form. Methionine concentration modified the cytotoxicity. Together, this indicates that selenomethionine uptake and enzymic metabolism are involved in the cytotoxicity in a yet unknown way.     

Prominent mitochondrial DNA recombination intermediates in human heart muscle

Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle. Replication intermediates from both the coupled and strand-asynchronous mtDNA replication pathways were detected. An additional class of non-linear molecules, with the electrophoretic properties of four-way junctions, was also prominent. These molecules were insensitive to topoisomerase I or RNase H, but were diminished by branch migration or RuvC treatment. Junctional molecules were detected in all regions of the mitochondrial genome, were found in myocardial DNA from young and old adults, but were present at lower levels in skeletal muscle and placenta. We suggest that they could represent intermediates of mtDNA repair, given their prevalence in the oxyradical-rich environment of heart muscle mitochondria.     

Crystal structure and role of glycans and dimerization in folding of neuronal leucine-rich repeat protein AMIGO-1

AMIGO-1 is the parent member of a novel family of three cell surface leucine-rich repeat (LRR) proteins. Its expression is induced by the binding of HMGB1 (high-mobility group box 1 protein) to RAGE (receptor for advanced glycation end products) on neurons. Binding of HMGB1 to RAGE is known to have a direct effect on cellular growth regulation and mobility, and AMIGO-1 directly supports growth of neuronal processes and fasciculation of neurites. In addition, the second member of the AMIGO-family, AMIGO-2, has been implicated in adhesion of tumor cells in adenocarcinoma and survival of neurons.We have determined the crystal structure of AMIGO-1 at 2.0 Å resolution, which reveals a typical cell surface LRR domain arrangement with N- and C-terminal capping domains with disulfide bridges, followed by a C2-type Ig domain. AMIGO-1 is a dimer, with the LRR regions forming the dimer interface, and sequence conservation analysis and static light-scattering measurements suggest that all three AMIGO family proteins form similar dimers. Based on the AMIGO-1 structure, we have also modeled AMIGO-2 and present small-angle X-ray scattering data on AMIGO-2 and AMIGO-3. Our mutagenesis studies show that AMIGO-1 dimerization is necessary for proper cell surface expression and thus probably for proper or stable folding in the endoplastic reticulum and for the function of the protein. Based on the data presented earlier, we also suggest that dimerization through the LRR-LRR interface is likely to be involved in cell-cell adhesion by AMIGO-1, while extensive glycosylation may have a role.     

Lifetable demography and population growth of the rotifer Brachionus angularis in Kenya: influence of temperature and food density

Lifetable demography and reproductive traits of a Kenyan strain of the rotifer Brachionus angularis were investigated using individual and small batch culture approaches. The rotifer was identified morphologically before conducting studies at 20, 25 and 30 °C, using Chlorella vulgaris at 2.5 × 10⁵ to 2.5 × 10⁷ cells ml^–1. The rotifers were highly fecund, producing 2.11 ± 0.07 offspring female^–1 day^–1 and reproductive, producing 8.43 ± 0.24 offspring female^–1 at 25 °C with 2.5 × 10⁶ algal cells ml^–1. The highest intrinsic rate of natural increase (0.74 ± 0.02 d^–1), specific population growth rate (0.49 ± 0.01), longest life expectancy at hatching (12.41 ± 0.28 d) and shortest generation time (2.87 ± 0.03 d) also occurred at 25 °C with 2.5 × 10⁶ algal cells ml^–1. The duration of hatching to first spawning was shortest (2.86 ± 0.21 h) at 30 °C with 2.5 × 10⁷ algal cells ml^–1 and longest (8.83 ± 0.39 h) at 20 °C with 2.5 × 10⁵ algal cells ml^–1. The highest population density (255.7 ± 12.6 ind. ml^–1) was realised at 25 °C with 2.5 × 10⁶ cells ml^–1 on Day 8, whereas the lowest population density (122.0 ± 3.6 ind. ml^–1) was realised at 20 °C with 2.5 × 10⁵ cells ml^–1 on Day 8. The lorica length and width of the Kenyan strain of B. angularis are 85.6 ± 3.1 µm and 75.4 ± 3.6 µm, respectively. The rotifer optimally reproduces at 25 °C when fed with 2.5 × 10⁶ algal cells ml^–1.     

Water level fluctuations in Lake Baringo,Kenya, during the 19th and 20th centuries: Evidence from lake sediments

This article presents a fossil diatom-based, semi-quantitative reconstruction of water level fluctuations for Lake Baringo over the past 200 years as a consequence of climatic variations. A 285 cm long sediment core sample was collected using a Rod-Operated Single-drive Stationary Piston corer. Lake level was inferred using indices based on the proportion of planktonic to benthic diatom taxa (P/B ratio). The sediment archive presented distinct zones dominated by planktonic and benthic diatom flora. An initial transgression in the early 19th century was characterised as a shallow water environment dominated by planktonic Aulacoseira spp. This was a response to extreme drought during the late 18th to early 19th century. Mid-19th century was defined by a high lake stand. The late 19th to early 20th centuries experienced low water level following the widely documented aridity at the time. The mid-20th century was marked by a spectacular rise in water level that coincided with remarkably wet years during the early 1960s and late 1970s. The first decade of the 21st century witnessed widespread changes in water level. The proxy records show that lake ramping and drawdown over the years follow approximately 50-year climatic cycles.     

Nanobacteria--propagating calcifying nanoparticles

Nanobacteria, also known as calcifying nanoparticles (CNP), are controversial infectious agents not matching the current criteria for 'living organism'. Despite the controversy of their classification, they propagate and cause cell death in vitro and are associated or found in many human diseases. Thus, more efforts should be focussed on research on pathogenicity of CNP.     

A new method for the assay of tissue. S-adenosylhomocysteine and S-adenosylmethione. Effect of pyridoxine deficiency on the metabolism of S-adenosylhomocysteine, S-adenosylmethionine and polyamines in rat liver.

The hepatic synthesis and accumulation of S-adenosylhomocysteine, S-adenosylmethionine and polyamines were studied in normal and vitamin B-6-deficient male albino rats. A method involving a single chromatography on a phosphocellulose column was developed for the determination of S-adenosylhomocysteine and S-adenosylmethionine from tissue samples. Feeding the rat with pyridoxine-deficient diet for 3 or 6 weeks resulted in a four- to five-fold increase in the concentration of S-adenosylhomocysteine, whereas that of S-adenosylmethionine was only slighly elevated. The concentration of putrescine was decreased to half, that of spermidine was somewhat decreased and that of spermine remained fairly constant. The activities of L-ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, L-methionine adenosyltransferase and S-adenosyl-L-homocysteine hydrolase were moderately increased. S-Adenosylmethionine decarboxylase showed no requirement for pyridoxal 5'-phosphate. The major effect of pyridoxine deficiency of S-adenosylmethionine metabolism seems to be a block in the utilization of S-adenosylhomocysteine, resulting in the accumulation of this metabolite to a concentration that may inhibit biological methylation reactions.     

Effects of selenomethionine on cell growth and on S-adenosylmethionine metabolism in cultured malignant cells.

The effects of selenomethionine (SeMet) on the growth of 17 cultured cell lines were studied. SeMet in the culture medium of three hepatoma cell lines promoted cell growth at subcytotoxic levels (1-20 microM), but the growth of malignant lymphoid and myeloid cells was not stimulated. L-SeMet was cytotoxic to all 17 cell lines when assayed after culture for 3-10 days. A 50% growth inhibition was observed by 30-160 microM-SeMet in a culture medium containing 100 microM-methionine. SeMet cytotoxicity to normal (fibroblasts) and malignant cells was rather similar, excluding specific antineoplastic cytotoxicity. Cytotoxicity was increased by decreasing concentrations of methionine. The DL form of SeMet was less cytotoxic than the L form. L-SeMet was metabolized to a selenium analogue of S-adenosylmethionine approximately as effectively as the natural sulphur analogue methionine in malignant R1.1 lymphoblasts. Concomitantly, S-adenosylmethionine pools were decreased. This occurred early and at cytotoxic SeMet levels. Methionine adenosyltransferase activity was not altered by SeMet treatment. ATP pools were not affected early, and decreases in the synthesis of DNA and protein took place late and were apparently related to cell death. RNA synthesis was slightly stimulated at low cytotoxic SeMet levels by 24 h, but was markedly inhibited after 48 h. The SeMet analogue of S-adenosylmethionine could be effectively utilized in a specific enzymic transmethylation. Neither S-adenosylhomocysteine nor its selenium analogue accumulated in the treated cells. These findings together suggest a direct or indirect involvement of S-adenosylmethionine metabolism in SeMet cytotoxicity, but exclude a gross blockage of transmethylations.     

Trophic state and nutrient limitation in Lake Baringo,Kenya

The trophic state of Lake Baringo and factors that could be limiting the development of algal biomass in it were investigated during one wet/dry hydrological cycle in 2014–2015. Water samples were analysed for dissolved inorganic nutrients, including , and , total phosphorus and Chlorophyll a. Light attenuation was estimated using Secchi depth. The trophic state was determined using Carlson trophic state indices (CTSI). Deviations in CTSI, nutrient ratios and ambient nutrient concentrations were used to identify factors limiting phytoplankton growth. The mean values measured for Secchi depth, nitrate, total phosphorus and Chlorophyll a showed significant seasonal variation (p < 0.05). Based on the Carlson trophic state index, the results show that Lake Baringo is eutrophic. However, the lake is also experiencing phosphorus limitation and poor light penetration, because of high turbidity, which is more pronounced during the wet season.