Combined lipase deficiency (cld/cld) in mice. Demonstration that an inactive form of lipoprotein lipase is synthesized

Combined lipase deficiency, cld, is a recessive mutation within the T/t complex of mouse chromosome 17. Mice homozygous for this defect display severe functional deficiencies of lipoprotein lipase and the related hepatic lipase. They develop massive hyperchylomicronemia and die within 3 days when allowed to suckle. Heart, diaphragm muscle, and brown adipose tissue of 1-day-old cld/cld and unaffected mice incorporated in vivo [35S]methionine into a protein that could be immunoprecipitated by antilipoprotein lipase serum. The immunoprecipitated protein in all tissues had the same Mr as bovine lipoprotein lipase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proportion of radioactivity in the lipoprotein lipase band to that in total protein was 0.02% in tissues of cld/cld mice and 0.01% in tissues of unaffected mice. There was 2-6 times more lipoprotein lipase-like protein (determined by immunoassay) in tissues of defective mice than in those of unaffected mice. These findings indicate that the cld mutation did not cause deletion of the structural gene for lipoprotein lipase. Lipoprotein lipase activity in heart, diaphragm muscle, brown adipose tissue, and lung of cld/cld mice was less than 5% of that in tissues of unaffected mice. This low activity could be inhibited more than 85% by antilipoprotein lipase serum, but not by nonimmune serum. It is concluded that tissues in cld/cld mice synthesize a lipoprotein lipase-like protein which has subnormal catalytic activity.     

Effect of epinephrine and other lipolytic agents on intracellular lipolysis and lipoprotein lipase activity in 3T3-L1 adipocytes

3T3-L1 adipocytes were used to test the hypothesis that hormone-sensitive lipolysis and lipoprotein lipase activity might be regulated in a reciprocal manner. Intracellular lipolysis was stimulated by catecholamine, dibutyryl cAMP, and ACTH, but not by glucagon. The effects of epinephrine on lipolysis were blocked by the beta-antagonist propanolol but not by the alpha-antagonist phentolamine. Hormone-stimulated lipolysis was not changed by acute (45 min) or chronic (2 days) treatment of the cells with insulin whereas the latter treatment augmented lipoprotein lipase activity about fivefold. Epinephrine did not affect the lipoprotein lipase activity of insulin-stimulated cells. Withdrawal of glucose from the medium decreased lipoprotein lipase activity and the effect of epinephrine on lipolysis. Effects of lipolytic agents on activity of lipoprotein lipase were variable and concentration-dependent. Lipoprotein lipase activity was decreased only by concentrations of epinephrine greater than those inducing maximal intracellular lipolysis, and the decrease in activity occurred about 30 min after the increase in glycerol release. There seems to be no relationship between the level of activity of lipoprotein lipase and the maximal rate of hormone-stimulated lipolysis in 3T3-L1 cells. Unlike in adipose tissue and adipocytes of rats, hormone-stimulated lipolysis and lipoprotein lipase activity in murine 3T3-L1 adipocytes appear to be regulated independently.     

A Biochemical Study of Spore Germination in the Blue-green Alga Anabaena doliolum

The spores of Anabaena doliolum formed in light (light spores)and after transfer to darkness (dark spores) are biochemicallydifferent in that the light spores contain chlorophyll a andphycocyanin, while dark spores seem to lack them. The apparentbiosyntheses accompanying dark-spore germination seem to proceedin the following order: RNA, chlorophyll a, phycocyanin andDNA. Results of chloramphenicol treatment indicate that proteinsynthesis precedes RNA synthesis. The biosynthetic events followingRNA synthesis show a requirement for light.     

EFFECTS OF LIPOPROTEIN LIPASE ON THE STRUCTURE OF CHYLOMICRONS

Chylomicrons isolated from rat lymph were complexed with lipoprotein lipase of post-heparin plasma (chylomicrons-LPL) in order to study the effects of lipolysis on the structure of chylomicrons. Triglyceride in the chylomicron core was readily hydrolyzed to free fatty acids (FFA) and glycerol when chylomicrons-LPL were incubated at pH 8.3 in medium containing albumin. Although most of the FFA were immediately released to the medium, some were retained within chylomicrons when FFA-binding sites on albumin were not available. These observations suggest that albumin may have a specific role in the transfer of FFA across the chylomicron surface film. Chylomicrons-LPL assumed many different shapes as they were depleted of triglyceride by the lipolytic action of the enzyme, and total removal of core triglyceride resulted in empty sacks of surface film. The surface film was visualized in sections of OsO₄-fixed chylomicrons-LPL as a thin electron-opaque line, 25–30 Å wide, in areas where the underlying electron-opaque core had been replaced by zones of decreased electron opacity, and in folds of surface film extending outward from chylomicrons partially depleted of core lipid. The findings demonstrate that chylomicrons consist of a core of liquid triglyceride enveloped by a pliable and durable monolayer surface film, and that lipoprotein lipase reduces the triglyceride core without disrupting the surface film.     

Synthesis of inactive nonsecretable high mannose-type lipoprotein lipase by cultured brown adipocytes of combined lipase-deficient cld/cld mice

Combined lipase deficiency (cld) is a recessive mutation which causes a severe deficiency of lipoprotein lipase and hepatic lipase activities and lethal hypertriacylglycerolemia within 3 days in newborn mice. The effect of this genetic defect on lipoprotein lipase was studied in primary cultures of brown adipocytes derived from tissue of newborn mice. Cells cultured from cld/cld mice replicated, accumulated triacylglycerol, and differentiated into adipocytes at normal rates. Lipoprotein lipase activity in unaffected cells was detectable on Day 0 of confluence and increased to 1.3 units/mg DNA by Day 6, while that in cld/cld cells was less than 4% of that in unaffected cells on Days 4-6. Unaffected cells released 1.2% of their lipase activity in 30 min in the absence of heparin, and 11% in 10 min in the presence of heparin, whereas cld/cld cells released no lipase activity. cld/cld cells contained 2-3 times as much lipoprotein lipase protein as unaffected cells, and released no lipase protein to the medium. Immunofluorescent lipoprotein lipase was not detectable in unaffected adipocytes unless lipase secretion was blocked with monesin, causing retention of the lipase in Golgi. cld/cld adipocytes, in contrast, contained immunofluorescent lipoprotein lipase distributed in a diffuse reticular pattern, indicating retention of lipase in endoplasmic reticulum. Lipoprotein lipase immunoprecipitated from cells incubated 1-3 h with [35S]methionine was digested with or without endoglycosidase H (endo H) or F, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lipoprotein lipase in unaffected cells (Mr = 56,000-58,000) consisted of three glycosylated forms, of which the most prevalent was endo H-resistant, the next was totally endo H-sensitive, and the least was partially endo H-sensitive. In contrast, lipoprotein lipase in cld/cld cells (Mr = 56,000) consisted of a single, totally endo H-sensitive form. Lipoprotein lipase in both groups of cells contained two oligosaccharide chains. Chromatography studies with heparin-Sepharose indicated that at least some of the lipoprotein lipase in cld/cld cells was dimerized. The findings demonstrate that brown adipocytes cultured from cld/cld mice synthesize lipoprotein lipase with two high mannose oligosaccharide chains, but it is inactive and retained in endoplasmic reticulum. Whether the cld mutation affects primarily processing of oligosaccharide chains of lipoprotein lipase in endoplasmic reticulum, transport of the lipase from the reticulum, or some other process, is to be resolved.     

Evolutionary transfer of ORF-containing group I introns between different subcellular compartments (chloroplast and mitochondrion)

We describe here a case of homologous introns containing homologous open reading frames (ORFs) that are inserted at the same site in the large subunit (LSU) rRNA gene of different organelles in distantly related organisms. We show that the chloroplast LSU rRNA gene of the green alga Chlamydomonas pallidostigmatica contains a group I intron (CpLSU.2) encoding a site-specific endonuclease (I-CpaI). This intron is inserted at the identical site (corresponding to position 1931-1932 of the Escherichia coli 23S rRNA sequence) as a group I intron (AcLSU.m1) in the mitochondrial LSU rRNA gene of the amoeboid protozoon Acanthamoeba castellanii. The CpLSU.2 intron displays a remarkable degree of nucleotide similarity in both primary sequence and secondary structure to the AcLSU.m1 intron; moreover, the Acanthamoeba intron contains an ORF in the same location within its secondary structure as the CpLSU.2 ORF and shares with it a strikingly high level of amino acid similarity (65%; 42% identity). A comprehensive survey of intron distribution at site 1931 of the chloroplast LSU rRNA gene reveals a rather restricted occurrence within the polyphyletic genus Chlamydomonas, with no evidence of this intron among a number of non- Chlamydomonad green algae surveyed, nor in land plants. A parallel survey of homologues of a previously described and similar intron/ORF pair (C. reinhardtii chloroplast CrLSU/A. castellanii mitochondrial AcLSU.m3) also shows a restricted occurrence of this intron (site 2593) among chloroplasts, although the intron distribution is somewhat broader than that observed at site 1931, with site-2593 introns appearing in several green algal branches outside of the Chlamydomonas lineage. The available data, while not definitive, are most consistent with a relatively recent horizontal transfer of both site-1931 and site- 2593 introns (and their contained ORFs) between the chloroplast of a Chlamydomonas-type organism and the mitochondrion of an Acanthamoeba- like organism, probably in the direction chloroplast to mitochondrion. The data also suggest that both introns could have been acquired in a single event.      

Aerobic MTBE biodegradation: an examination of past studies, current challenges and future research directions

With the current practice of amending gasoline with up to 15% by volume MTBE, the contamination of groundwater by MTBE has become widespread. As a result, the bioremediation of MTBE-impacted aquifers has become an active area of research. A review of the current literature on the aerobic biodegradation of MTBE reveals that a number of cultures from diverse environments can either partially degrade or completely mineralize MTBE. MTBE is either utilized as a sole carbon and energy source or is degraded cometabolically by cultures grown on alkanes. Reported degradation rates range from 0.3 to 50 mg MTBE/g cells/h while growth rates (0.01–0.05 g MTBE/g cells/d) and cellular yields (0.1–0.2 g cells/g MTBE) are generally low. Studies on the mechanisms of MTBE degradation indicate that a monooxygenase enzyme cleaves the ether bond yielding tert-butyl alcohol (TBA) and formaldehyde as the dominant detectable intermediates. TBA is further degraded to 2-methyl-2-hydroxy-1-propanol, 2-hydroxyisobutyric acid, 2-propanol, acetone, hydroxyacteone and eventually, carbon dioxide. The majority of these intermediates are also common to mammalian MTBE metabolism. Laboratory studies on the degradation of MTBE in the presence of gasoline aromatics reveal that while degradation rates of other gasoline components are generally not inhibited by MTBE, MTBE degradation could be inhibited in the presence of more easily biodegradable compounds. Controlled field studies are clearly needed to elucidate MTBE degradation potential in co-contaminant plumes. Based on the reviewed studies, it is likely that a bioremediation strategy involving direct metabolism, cometabolism, bioaugmentation, or some combination thereof, could be applied as a feasible and cost-effective treatment method for MTBE contamination.     

Application of real-time PCR to study effects of ammonium on population size of ammonia-oxidizing bacteria in soil

Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammonia-monooxygenase gene (amoA) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 x 10(5) cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH4)2SO4 application. AOB populations in amended treatments increased from an initial density of approximately 4 x 10(6) cells/g of dry soil to peak values (day 7) of 35 x 10(6) and 66 x 10(6) cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 x 10(9) and 2.2 x 10(9) cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed in microcosms could also be detected in the field. AOB population size increased from 8.9 x 10(6) to 38.0 x 10(6) cells/g of soil by day 39. Generation times were 28 and 52 h in the 1.5 and 7.5 mM treatments, respectively, in the microcosm experiment and 373 h in the ammonium treatment in the field study. Estimated oxidation rates per cell ranged initially from 0.5 to 25.0 fmol of NH4+ h(-1) cell(-1) and decreased with time in both microcosms and the field. Growth yields were 5.6 x 10(6), 17.5 x 10(6), and 1.7 x 10(6) cells/mol of NH4+ in the 1.5 and 7.5 mM microcosm treatments and the field study, respectively. In a second field experiment, AOB population size was significantly greater in annually fertilized versus unfertilized soil, even though the last ammonium application occurred 8 months prior to measurement, suggesting a long-term effect of ammonium fertilization on AOB population size.     

Sediment microbial community structure and mercury methylation in mercury-polluted Clear Lake, California

Spatial and temporal variations in sediment microbial community structure in a eutrophic lake polluted with inorganic mercury were identified using polar lipid fatty acid (PLFA) analysis. Microbial community structure was strongly related to mercury methylation potential, sediment organic carbon content, and lake location. Pore water sulfate, total mercury concentrations, and organic matter C/N ratios showed no relationships with microbial community structure. Seasonal changes and changes potentially attributable to temperature regulation of bacterial membranes were detectable but were less important influences on sediment PLFA composition than were differences due to lake sampling location. Analysis of biomarker PLFAs characteristic of Desulfobacter and Desulfovibrio groups of sulfate-reducing bacteria suggests that Desulfobacter-like organisms are important mercury methylators in the sediments, especially in the Lower Arm of Clear Lake.     

Changes in Soil Microbial Community Structure Influenced by Agricultural Management Practices in a Mediterranean Agro-Ecosystem

Agricultural practices have proven to be unsuitable in many cases, causing considerable reductions in soil quality. Land management practices can provide solutions to this problem and contribute to get a sustainable agriculture model. The main objective of this work was to assess the effect of different agricultural management practices on soil microbial community structure (evaluated as abundance of phospholipid fatty acids, PLFA). Five different treatments were selected, based on the most common practices used by farmers in the study area (eastern Spain): residual herbicides, tillage, tillage with oats and oats straw mulching; these agricultural practices were evaluated against an abandoned land after farming and an adjacent long term wild forest coverage. The results showed a substantial level of differentiation in the microbial community structure, in terms of management practices, which was highly associated with soil organic matter content. Addition of oats straw led to a microbial community structure closer to wild forest coverage soil, associated with increases in organic carbon, microbial biomass and fungal abundances. The microbial community composition of the abandoned agricultural soil was characterised by increases in both fungal abundances and the metabolic quotient (soil respiration per unit of microbial biomass), suggesting an increase in the stability of organic carbon. The ratio of bacteria:fungi was higher in wild forest coverage and land abandoned systems, as well as in the soil treated with oat straw. The most intensively managed soils showed higher abundances of bacteria and actinobacteria. Thus, the application of organic matter, such as oats straw, appears to be a sustainable management practice that enhances organic carbon, microbial biomass and activity and fungal abundances, thereby changing the microbial community structure to one more similar to those observed in soils under wild forest coverage.