Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine

One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.     

LETM1, a gene deleted in Wolf-Hirschhorn syndrome, encodes an evolutionarily conserved mitochondrial protein

The leucine zipper-, EF-hand-containing transmembrane protein 1 (LETM1) has recently been cloned in an attempt to identify genes deleted in Wolf-Hirschhorn syndrome (WHS), a microdeletion syndrome characterized by severe growth and mental retardation, hypotonia, seizures, and typical facial dysmorphic features. LETM1 is deleted in almost all patients with the full phenotype and has recently been suggested as an excellent candidate gene for the seizures in WHS patients. We have shown that LETM1 is evolutionarily conserved throughout the eukaryotic kingdom and exhibits homology to MDM38, a putative yeast protein involved in mitochondrial morphology. Using LETM1-EGFP fusion constructs and an anti-rat LetM1 polyclonal antibody we have demonstrated that LETM1 is located in the mitochondria. The present study presents information about a possible function for LETM1 and suggests that at least some (neuromuscular) features of WHS may be caused by mitochondrial dysfunction.     

Molecular analysis in patients with mucopolysaccharidosis type II suggests that DXS466 maps within the Hunter gene

Hunter disease is an X-linked mucopolysaccharidosis caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Using the IDS cDNA and DNA probes corresponding to loci flanking the IDS locus, we performed molecular genetic studies in two patients with Hunter syndrome. An interstitial deletion spanning the middle part of the IDS gene was found in the first patient. The second patient carries a gross gene rearrangement that can be detected after HindIII or EcoRI digestion of genomic DNA, and is similar to that found recently in seven unrelated Hunter patients. Our data suggest that the structural aberration observed is a partial intragenic inversion. As the same altered hybridization pattern is also revealed by the recently described anonymous DNA probe II 10, which recognizes a frequent TaqI restriction fragment length polymorphism at the DXS466 locus, we conclude that DXS466 maps within the IDS gene, probably in an intron.     

From left to right all through the night: Characteristics of lying rest in zoo elephants

Despite increased research during the past years, many characteristics of resting behavior in elephants are still unknown. For example, there is only limited data suggesting elephants express longer lying bouts and increased total nightly lying durations on soft substrates as compared to hard surfaces. Additionally, it has not been investigated how frequently elephants change body sides between lying bouts. Here we present these characteristics based on observations of nighttime lying behavior in 10 zoo elephants (5 African Loxodonta africana and 5 Asian Elephas maximus elephants) living in five different European facilities. We found that elephants housed on soft substrates have significantly increased total lying durations per night and longer average lying bouts. Furthermore, at 70%−85% of all bouts, a consistently higher frequency of side change between lying bouts occurred on soft substrates, leading to an overall equal laterality in resting behavior. Deviations from this pattern became evident in elephants living on nonsand flooring or/and in nondominant individuals of nonfamily groups, respectively. Based on our findings, we consider elephants to normally have several lying bouts per night with frequent side changes, given an appropriate substrate and healthy social environment. We encourage elephant-keeping facilities to monitor these characteristics in their elephants' nighttime behavior to determine opportunities for further improvements and detect alterations putatively indicating social or health problems in individual elephants at an early stage.     

Hedgehog and retinoid signalling confines nkx2.2b expression to the lateral floor plate of the zebrafish trunk

The ventral neural tube of vertebrates consists of distinct neural progenitor domains positioned along the dorsoventral (DV) axis that develop different types of moto- and interneurons. Several signalling molecules, most notably Sonic Hedgehog (Shh), retinoic acid (RA) and fibroblast growth factor (FGF) have been implicated in the generation of these domains. Shh is secreted from the floor plate, the ventral most neural tube structure that consists of the medial (MFP) and the lateral floor plate (LFP). While the MFP is well characterized, organization and function of the LFP remains unclear. Here, we describe the novel homeobox gene nkx2.2b that is strongly expressed in the trunk LFP of zebrafish and thus represents a unique marker for the characterization of LFP formation and the identification of LFP deficient mutants. nkx2.2b and its paralog nkx2.2a (formerly known as nk2.2 and nkx2.2) arose by gene duplication in zebrafish. Both duplicates show significant differences in their expression patterns. For example, while prominent nkx2.2a expression has been described in the ventral brain [Barth, K.A., Wilson, S.W., 1995. Expression of zebrafish nk2.2 is influenced by sonic hedgehog/vertebrate hedgehog-1 and demarcates a zone of neuronal differentiation in the embryonic forebrain. Development 121, 1755-1768], hardly any expression can be found in the trunk LFP, which is in contrast to nkx2.2b. Overexpression, mutant and inhibitor analyses show that nkx2.2b expression in the LFP is up-regulated by Shh, but repressed by retinoids and ectopic islet-1 (isl1) expression. In contrast to previously described zebrafish trunk LFP markers, like e.g. tal2 or foxa2, nkx2.2b is exclusively expressed in the LFP. Thus, it represents a unique tool to analyse the mechanisms of ventral neural tube patterning in zebrafish.     

Imaging morphological details and pathological differences of red blood cells using tapping-mode AFM

The surface topography of red blood cells (RBCs) was investigated under near-physiological conditions using atomic force microscopy (AFM). An immobilization protocol was established where RBCs are coupled via molecular bonds of the membrane glycoproteins to wheat germ agglutinin (WGA), which is covalently and flexibly tethered to the support. This results in a tight but non-invasive attachment of the cells. Using tapping-mode AFM, which is known as gentle imaging mode and therefore most appropriate for soft biological samples like erythrocytes, it was possible to resolve membrane skeleton structures without major distortions or deformations of the cell surface. Significant differences in the morphology of RBCs from healthy humans and patients with systemic lupus erythematosus (SLE) were observed on topographical images. The surface of RBCs from SLE patients showed characteristic circular-shaped holes with approx. 200 nm in diameter under physiological conditions, a possible morphological correlate to previously published changes in the SLE erythrocyte membrane.     

Methane Emission by Camelids

Methane emissions from ruminant livestock have been intensively studied in order to reduce contribution to the greenhouse effect. Ruminants were found to produce more enteric methane than other mammalian herbivores. As camelids share some features of their digestive anatomy and physiology with ruminants, it has been proposed that they produce similar amounts of methane per unit of body mass. This is of special relevance for countrywide greenhouse gas budgets of countries that harbor large populations of camelids like Australia. However, hardly any quantitative methane emission measurements have been performed in camelids. In order to fill this gap, we carried out respiration chamber measurements with three camelid species (Vicugna pacos, Lama glama, Camelus bactrianus; n = 16 in total), all kept on a diet consisting of food produced from alfalfa only. The camelids produced less methane expressed on the basis of body mass (0.32±0.11 L kg⁻¹ d⁻¹) when compared to literature data on domestic ruminants fed on roughage diets (0.58±0.16 L kg⁻¹ d⁻¹). However, there was no significant difference between the two suborders when methane emission was expressed on the basis of digestible neutral detergent fiber intake (92.7±33.9 L kg⁻¹ in camelids vs. 86.2±12.1 L kg⁻¹ in ruminants). This implies that the pathways of methanogenesis forming part of the microbial digestion of fiber in the foregut are similar between the groups, and that the lower methane emission of camelids can be explained by their generally lower relative food intake. Our results suggest that the methane emission of Australia''s feral camels corresponds only to 1 to 2% of the methane amount produced by the countries'' domestic ruminants and that calculations of greenhouse gas budgets of countries with large camelid populations based on equations developed for ruminants are generally overestimating the actual levels.     

Blm10 facilitates nuclear import of proteasome core particles

Short‐lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEAT‐like modules and is structurally related to the nuclear import receptor importin/karyopherin β. In proliferating yeast, RP‐CP assemblies are primarily nuclear and promote cell division. During quiescence, RP‐CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10‐bound CP serves as an import receptor–cargo complex, as Blm10 mediates the interaction with FG‐rich nucleoporins and is dissociated from the CP by Ran‐GTP. Thus, Blm10 represents the first CP‐dedicated nuclear import receptor in yeast.