Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice

An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the in vitro data, deletion of FAAH lowered lyso-sulfatide levels in a mouse model of MLD. Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.     

Response of different decomposer communities to the manipulation of moisture availability: potential effects of changing precipitation patterns

The potential impacts of changes in precipitation patterns associated with global climate change on the relationship between soil community diversity and litter decomposition were investigated. For a period of ca. 5 months, two decomposer communities in litterbags (1000 and 45 μm mesh size) containing spruce litter were subjected to two irrigation treatments: constant and fluctuating (drying/rewetting) moisture conditions. The latter were expected to induce moisture stress on the decomposer communities. The two mesh sizes were used to exclude different faunal components from the decomposer communities. The 1000 μm mesh excluded only the macrofauna, whereas the 45 μm mesh excluded both the macro- and mesofauna. In the short-term perspective of the present study, mesofauna abundance showed no response to imposed fluctuating moisture conditions. Irrespective of the presence of mesofauna, mass loss, microbial biomass and the control mechanisms, regulating carbon mineralization appeared unaffected by fluctuating moisture conditions. The reduction in the functional/structural diversity of the decomposer communities in the 45 μm litterbags resulted in strongly increased Nematoda abundance but it did not alter the response of Nematoda to fluctuating moisture conditions. Processes in the nitrogen (N)-cycle and mass loss were sensitive indicators of changes in the structural and functional complexity of decomposer communities. However, a negative effect of fluctuating moisture conditions on extractable N was coupled to the presence of mesofauna. Extremes in rainfall patterns, generated by climate change, may have a negative impact on the availability of nutrients, particularly N, for plants. This effect could be amplified by an additional impoverishment in the structural and functional complexity of the respective decomposer communities.     

Accumulation of sulfatide in neuronal and glial cells of arylsulfatase A deficient mice

Arylsulfatase A (ASA) degrades sulfatide, seminolipid and lactosylceramide sulfate, glycolipids recognized by the Sulph I antibody although sulfatide is considered the main antigen. Sulfatide is myelin associated but studies have shown a minor distribution also in non-myelin forming cells. The aim of this work was to further study sulfatide in neurons and astrocytes by immunohistochemistry, facilitated by investigation of tissue from adult ASA deficient (ASA ?/?) mice. Cells with a low presence of sulfatide might be detected due to lack of ASA activity and accumulation of Sulph I antigens. Sulfatide positive astrocytes and neurons were more numerous and intensely stained in ASA ?/? mice, demonstrating a sulfatide accumulation compared to controls. Sulph I staining was especially increased in the molecular layer of cerebellum, in which Purkinje cell dendrites displayed an altered morphology, and in layer IV–VI of cerebral cortex. In hippocampus, immunostaining was found in neuronal cytoplasm in ASA ?/? but in nuclear membranes of control mice. We observed a gray matter astrogliosis, which appeared to be associated to sulfatide accumulation. In addition, the developmental change (<20 months) of Sulph I antigens, galactosylceramide, phospholipids and cholesterol were followed by lipid analyses which verified sulfatide and seminolipid accumulation in adult ASA ?/? mice, although no lactosylceramide sulfate could be detected. In addition to demonstrating sulfatide in neurons and astrocytes, this study supports the value of ASA ?/? mice as a model for metachromatic leukodystrophy and suggests that accumulation of sulfatide beyond myelin might contribute to the pathology of this disease.     

On Animal Abundance Estimation Based on Pitfall Traps

This paper gives a simple proof of an equation for the mean number of animals from a very large population which are trapped in a pitfall trap. This equation can be applied to estimate the abundance of epigeic animals walking on the ground such as some insects or spiders. For the estimation, knowledge on the locomotion behaviour of the animals is necessary. As an alternative, an approach is discussed that is based on the two‐dimensional Brownian motion.     

Synthesis and mesomorphic properties of glycosyl dialkyl- and diacyl-glycerols bearing saturated, unsaturated and methyl branched fatty acid and fatty alcohol chains. Part II. Mesomorphic properties

The biophysical properties of a series of glycosyl dialkyl- and diacyl-glycerols bearing unsaturated or chiral methyl branched chains in the tail, and di- and trisaccharide carbohydrate headgroups are described. Thermotropism was investigated by polarising microscopy, the lyotropism was investigated by small angle X-ray diffraction and by the contact preparation method, and the gel to liquid crystalline phase transition by FT-IR-spectroscopy. The compounds displayed thermotropic Smectic A (SmA), cubic and columnar phases, whereas in the lyotropic phase diagram lamellar, hexagonal and cubic phases are found. The introduction of unsaturated or methyl branched chains leads to liquid crystallinity at ambient temperature. The difference between the 1,3-oleyl-glycerol maltoside and the corresponding 1,2-oleoyl-glycerol maltoside is small.     

The TonB-dependent ferrichrome receptor FcuA of Yersinia enterocolitica: evidence against a strict co-evolution of receptor structure and substrate specificity

A Yersinia enterocolitica receptor mutant was isolated which is impaired in ferrichrome uptake. The receptor-encoding gene fcuA was cloned in Escherichia coli K-12. A fcuA mutant of Y. enterocolitica could be complemented by the cloned DNA fragment. The FcuA-encoding region was sequenced and an open reading frame encoding 758 amino acids including a signal sequence of 36 amino acids was found. FcuA shared 34.6% amino acid sequence homology with FatA, the anguibactin receptor of Vibrio anguillarum, but only 20.6% homology with FhuA, the ferrichrome receptor of E. coli Since the structure of anguibactin differs strongly from that of ferrichrome there seems to be no co-evolution of receptor structure and substrate specificity. The ferrichrome receptors FcuA from Y. enterocolitica and FhuA from E. coli had slightly different substrate specificities. In contrast to FhuA from E. coli, FcuA from Y. enterocolitica was more stereoselective and failed to transport enantio ferrichrome. Three additional ferrichrome receptors were cloned from Pantoea aggiomerans (formerly Erwinia herbicola), Salmonella paratyphi B and Salmonella typhimurium. Their substrate specificity was similar but not identical.     

An assay for the rapid detection of the arylsulfatase A pseudodeficiency allele facilitates diagnosis and genetic counseling for metachromatic leukodystrophy

Summary Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulfatase A (ASA). A substantial ASA deficiency has also been described in clinically healthy persons, a condition for which the term pseudodeficiency was introduced. The discrimination of both kinds of deficiencies based on ASA activity determination is difficult and unreliable. This creates a serious problem in the genetic counseling and diagnosis of MLD. The mutations characteristic for the pseudodeficiency (PD) allele have recently been identified. A non-radioactive assay based on the polymerase chain reaction is described, which allows the rapid detection of the ASA pd allele. The assay utilizes pairs of primers that allow either the amplification of the ASA PD allele or of other ASA alleles, since their 3 residues match either the ASA PD allele or other ASA alleles.     

Export and activity of hybrid FhuA''-''Iut receptor proteins and of truncated FhuA'' proteins of the outer membrane of Escherichia coli

Summary The FhuA protein in the outer membrane of Escherichia coli serves as a multifunctional receptor for the phages T5, T1, 80, for colicin M, for ferrichrome (Fe³⁺-siderophore) and for the structurally related antibiotic, albomycin. To determine structural domains required for these receptor functions and for export, a fusion protein between FhuA and Iut (receptor for Fe³⁺-aerobactin and cloacin DF13) was constructed. In the FhuA-Iut hybrid protein, 24 amino acids of FhuA were replaced by 19 amino acids, 18 of which were from Iut. The number of plaque forming units of phage T5 and T1 on cells expressing FhuA-Iut was nearly as high as on cells expressing plasmid-encoded wild-type FhuA. However, 10⁷-fold higher concentrations of phage 80 and 10³ times more colicin M were required to obtain a zone of growth inhibition. Truncated FhuA proteins in which the last 24 amino acids at the carboxy-terminus were replaced by 16 (FhuA2) or 3 (FhuAT) amino acids could hardly be detected on polyacrylamide electrophoretograms of outer membrane proteins, due to proteolytic degradation. Sensitivity of cells expressing FhuA2 to phage T5 and T1 was reduced by several orders of magnitude and sensitivity to phage 80 and colicin M was totally abolished. In contrast, cells expressing FhuAT were nearly as sensitive to phage T5, T1, and 80 and to colicin M as cells containing FhuA-Iut. None of the constructs could grow on ferrichrome as sole iron source and none was sensitive to albomycin. Ferrichrome did not inhibit binding of T5 to TonB^– cells expressing FhuA-Iut (as it did in FhuA⁺ cells) due to the lack of ferrichrome binding. It is concluded that very small deletions (relative to the size of FhuA with 714 amino acids) at the C-terminal end render FhuA susceptible to proteolytic cleavage. The C-terminal alterations affect sensitivity to FhuA-specific agents very differently. Apparently, the C-terminus is a very important part of FhuA regarding stability and activity. Expression of active FhuA and partially inactive FhuA derivatives in the same cell revealed no negative complementation, suggesting a FhuA monomer as functional unit.