Anaerobic oxidation of methane in sediments of Lake Constance, an oligotrophic freshwater lake

Anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor has been reported for various environments, including freshwater habitats, and also, nitrate and nitrite were recently shown to act as electron acceptors for methane oxidation in eutrophic freshwater habitats. Radiotracer experiments with sediment material of Lake Constance, an oligotrophic freshwater lake, were performed to follow 14CO2 formation from 14CH4 in sediment incubations in the presence of different electron acceptors, namely, nitrate, nitrite, sulfate, or oxygen. Whereas 14CO2 formation without and with sulfate addition was negligible, addition of nitrate increased 14CO2 formation significantly, suggesting that AOM could be coupled to denitrification. Nonetheless, denitrification-dependent AOM rates remained at least 1 order of magnitude lower than rates of aerobic methane oxidation. Using molecular techniques, putative denitrifying methanotrophs belonging to the NC10 phylum were detected on the basis of the pmoA and 16S rRNA gene sequences. These findings show that sulfate-dependent AOM was insignificant in Lake constant sediments. However, AOM can also be coupled to denitrification in this oligotrophic freshwater habitat, providing first indications that this might be a widespread process that plays an important role in mitigating methane emissions.     

An experimentally validated genome-scale metabolic reconstruction of Klebsiella pneumoniae MGH 78578, iYL1228

Klebsiella pneumoniae is a Gram-negative bacterium of the family Enterobacteriaceae that possesses diverse metabolic capabilities: many strains are leading causes of hospital-acquired infections that are often refractory to multiple antibiotics, yet other strains are metabolically engineered and used for production of commercially valuable chemicals. To study its metabolism, we constructed a genome-scale metabolic model (iYL1228) for strain MGH 78578, experimentally determined its biomass composition, experimentally determined its ability to grow on a broad range of carbon, nitrogen, phosphorus and sulfur sources, and assessed the ability of the model to accurately simulate growth versus no growth on these substrates. The model contains 1,228 genes encoding 1,188 enzymes that catalyze 1,970 reactions and accurately simulates growth on 84% of the substrates tested. Furthermore, quantitative comparison of growth rates between the model and experimental data for nine of the substrates also showed good agreement. The genome-scale metabolic reconstruction for K. pneumoniae presented here thus provides an experimentally validated in silico platform for further studies of this important industrial and biomedical organism.     

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.     

Reduced Activity of Geranylgeranyl Reductase Leads to Loss of Chlorophyll and Tocopherol and to Partially Geranylgeranylated Chlorophyll in Transgenic Tobacco Plants Expressing Antisense RNA for Geranylgeranyl Reductase

The enzyme geranylgeranyl reductase (CHL P) catalyzes the reduction of geranylgeranyl diphosphate to phytyl diphosphate. We identified a tobacco (Nicotiana tabacum) cDNA sequence encoding a 52-kD precursor protein homologous to the Arabidopsis and bacterial CHL P. The effects of deficient CHL P activity on chlorophyll (Chl) and tocopherol contents were studied in transgenic plants expressing antisense CHL P RNA. Transformants with gradually reduced Chl P expression showed a delayed growth rate and a pale or variegated phenotype. Transformants grown in high (500 μmol m⁻² s⁻¹; HL) and low (70 μmol photon m⁻² s⁻¹; LL) light displayed a similar degree of reduced tocopherol content during leaf development, although growth of wild-type plants in HL conditions led to up to a 2-fold increase in tocopherol content. The total Chl content was more rapidly reduced during HL than LL conditions. Up to 58% of the Chl content was esterified with geranylgeraniol instead of phytol under LL conditions. Our results indicate that CHL P provides phytol for both tocopherol and Chl synthesis. The transformants are a valuable model with which to investigate the adaptation of plants with modified tocopherol levels against deleterious environmental conditions.     

Independent hybrid populations of <Emphasis Type="Italic">Formica polyctena X</Emphasis><Emphasis Type="Italic">rufa</Emphasis> wood ants (Hymenoptera: Formicidae) abound under conditions of forest fragmentation

Combined genetic and morphological data indicate frequent hybridisation between the wood ants Formica polyctena Förster 1850 and F. rufa Linnaeus 1761 in Central Europe. The genetic and morphological traits give a concordant picture of hybridisation with a strong correlation between the genotypic admixture proportions at 19 microsatellite loci and the first vectors of a principal component analysis (P < 0.001) and of a 3-class discriminant analysis (P < 0.001) of 15 quantitative morphological characters. This integrative approach enabled a grouping into F. polyctena, the hybrid and rufa. Genetic differentiation between the hybrid and F. rufa is significantly larger than between the hybrid and polyctena, indicating gene flow mainly between the latter entities. A suggested gene flow bias towards F. polyctena agrees with differential queen acceptance and mating behaviour. Both genetic and phenotypic colony parameters indicate predominance of monogyny in F. rufa but of polygyny in polyctena and the hybrid. Hybrids are intermediate between the parental species in body size, diagnostic morphological characters, monogyny frequency, size of nest population, nest diameter and infestation rate with epizootic fungi. The three entities respond differently to woodland fragmentation. Hybrids are significantly more abundant in forests with a coherent area <300 ha than in woodland above this size. Regions with high hybrid frequency in Germany—the Eastern Oberlausitz (23%) and the Baltic Sea islands Darss, Hiddensee and Rügen (28%)—are characterised by a fragmented woodland structure whereas regions with low hybrid frequency—Brandenburg and the lower Erzgebirge (3.4%)—have clearly larger and more coherent forest systems. Data from other European countries indicate habitat fragmentation to be a facilitating factor but no essential precondition for interspecific hybridisation in these ants. Hybrids are hypothesised to have selective advantage in fragmented systems because of combining the main reproductive and dispersal strategies of the parental species.     

Homophilic adhesion and CEACAM1-S regulate dimerization of CEACAM1-L and recruitment of SHP-2 and c-Src

Carcinoembryonic antigen (CEA)–related cell adhesion molecule 1 (CAM1 [CEACAM1]) mediates homophilic cell adhesion and regulates signaling. Although there is evidence that CEACAM1 binds and activates SHP-1, SHP-2, and c-Src, knowledge about the mechanism of transmembrane signaling is lacking. To analyze the regulation of SHP-1/SHP-2/c-Src binding, we expressed various CFP/YFP-tagged CEACAM1 isoforms in epithelial cells. The supramolecular organization of CEACAM1 was examined by cross-linking, coclustering, coimmunoprecipitation, and fluorescence resonance energy transfer. SHP-1/SHP-2/c-Src binding was monitored by coimmunoprecipitation and phosphotyrosine-induced recruitment to CEACAM1-L in cellular monolayers. We find that trans-homophilic CEACAM1 binding induces cis-dimerization by an allosteric mechanism transmitted by the N-terminal immunoglobulin-like domain. The balance of SHP-2 and c-Src binding is dependent on the monomer/dimer equilibrium of CEACAM1-L and is regulated by trans-binding, whereas SHP-1 does not bind under physiological conditions. CEACAM1-L homodimer formation is reduced by coexpression of CEACAM1-S and modulated by antibody ligation. These data suggest that transmembrane signaling by CEACAM1 operates by alteration of the monomer/dimer equilibrium, which leads to changes in the SHP-2/c-Src–binding ratio.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse,a model of Huntington’s disease

Huntington’s disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor–based Ca²⁺ signaling, which is crucial for skeletal muscle excitation–contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca²⁺ transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca²⁺ inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca²⁺ transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca²⁺ removal from the myoplasm and Ca²⁺ release flux from the sarcoplasmic reticulum were characterized using a Ca²⁺ binding and transport model, which indicated a significant reduction in slow Ca²⁺ removal activity and Ca²⁺ release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca²⁺ channel conductance. These results indicate profound changes of Ca²⁺ turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD.