Linkage of X-linked retinitis pigmentosa to the hypervariable DNA marker M27β (DXS255)

Summary A hypervariable DNA marker is closely linked to one of the most severe forms of night blindness, X-linked retinitis pigmentosa (RP). Affected individuals with X-linked RP, obligate carriers, and ophthalmologically identifiable carriers of the disease were included in a linkage study. The diagnosis was established in five sibships by funduscopic and electrophysiological investigations. When the X-linked probe M27 was used, 2 recombinants out of 29 informative meioses were detected (=0.07 at a maximum lod of 4.75). The hypervariable probe detected two different alleles in 38 of 39 females tested. M27 is therefore a potentially very useful probe for carrier detection and prenatal diagnosis, as well as for addressing the question of heterogeneity of X-linked RP.     

Proteomic identification and characterization of secreted N‐glycosylated NPC2 following cross‐linking of the high‐affinity receptor for IgE on mast cells

Allergen‐mediated cross‐linking of the high‐affinity receptor for IgE on mast cells triggers the release of diverse preformed and de novo synthesized immunoregulatory mediators that further the allergic response. A proteomic screen applied to the detection of proteins secreted by the model rat mast cell line, RBL‐2H3 (rat basophilic leukaemia, subline 2H3.1), led to the identification of the cholesterol‐binding glycoprotein, NPC2/RE1 (Niemann–Pick Type C2/epididymal secretory protein 1). Glycosylated NPC2 is secreted early in response to an IgE‐mediated stimulus and co‐localizes with the lysosomal membrane marker, CD63. NPC2 belongs to the ML (MD‐2‐related lipid‐recognition) protein family (155 members), which includes the Toll‐like receptor co‐factors, MD‐1 and MD‐2, and perhaps most interestingly, seven major house dust mite allergens of unknown function (including Der p 2 and Der f 2). Possible role(s) for the protein in the allergic response and future applications of this approach are discussed.     

3-Hydroxypropionyl-Coenzyme A Dehydratase and Acryloyl-Coenzyme A Reductase,Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in the Sulfolobales

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO₂ fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.In the thermoacidophilic autotrophic crenarchaeum Metallosphaera sedula, CO₂ fixation proceeds via a 3-hydroxypropionate/4-hydroxybutyrate cycle (8, 23, 24, 28) (Fig. ​(Fig.1).1). A similar cycle may operate in other autotrophic members of the Sulfolobales and in mesophilic Crenarchaea (Cenarchaeum sp. and Nitrosopumilus sp.) of marine group I. The cycle uses elements of the 3-hydroxypropionate cycle that was originally discovered in the phototrophic bacterium Chloroflexus aurantiacus (11, 16, 17, 19, 20, 32, 33). It involves the carboxylation of acetyl-coenzyme A (CoA) to malonyl-CoA by the biotin-dependent acetyl-CoA carboxylase. Malonyl-CoA is reduced via malonate semialdehyde to 3-hydroxypropionate (1), which is further reductively converted to propionyl-CoA (3). Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by a propionyl-CoA carboxylase that is similar or identical to acetyl-CoA carboxylase. In fact, only one copy of the genes for the acetyl-CoA/propionyl-CoA carboxylase subunits is present in most Archaea, suggesting that this is a promiscuous enzyme that acts on both acetyl-CoA and propionyl-CoA (24). (S)-Methylmalonyl-CoA is epimerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula and other members of the Sulfolobales. Enzymes are the following: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonate semialdehyde reductase (NADPH); 4, 3-hydroxypropionyl-CoA synthetase (3-hydroxypropionate-CoA ligase, AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinate semialdehyde reductase (NADPH); 12, 4-hydroxybutyryl-CoA synthetase (4-hydroxybutyrate-CoA ligase, AMP-forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14, crotonyl-CoA hydratase; 15, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase. The two steps of interest are highlighted.In Chloroflexus succinyl-CoA is converted to (S)-malyl-CoA, which is cleaved by (S)-malyl-CoA lyase to acetyl-CoA (thus regenerating the CO₂ acceptor molecule) and glyoxylate (16). Glyoxylate is assimilated into cell material by a yet not completely resolved pathway (37). In Metallosphaera succinyl-CoA is converted via 4-hydroxybutyrate to two molecules of acetyl-CoA (8), thus regenerating the starting CO₂ acceptor molecule and releasing another acetyl-CoA for biosynthesis. Hence, the 3-hydroxypropionate/4-hydroxybutyrate cycle (Fig. ​(Fig.1)1) can be divided into two parts. The first part transforms one acetyl-CoA and two bicarbonates into succinyl-CoA, and the second part converts succinyl-CoA to two acetyl-CoA molecules.The reductive conversion of 3-hydroxypropionate to propionyl-CoA requires three enzymatic steps: activation of 3-hydroxypropionate to its CoA ester, dehydration of 3-hydroxypropionyl-CoA to acryloyl-CoA, and reduction of acryloyl-CoA to propionyl-CoA. In C. aurantiacus these three steps are catalyzed by a single large trifunctional enzyme, propionyl-CoA synthase (2). This 200-kDa fusion protein consists of a CoA ligase, a dehydratase, and a reductase domain. Attempts to isolate a similar enzyme from M. sedula failed. Rather, a 3-hydroxypropionyl-CoA synthetase was found (3), suggesting that the other two reactions may also be catalyzed by individual enzymes.Here, we purified the missing enzymes 3-hydroxypropionyl-CoA dehydratase and acryloyl-CoA reductase from M. sedula, identified the coding genes in the genome of M. sedula and other members of the Sulfolobales, produced recombinant enzymes as proof of function, and studied the enzymes in some detail. A comparison with the respective domains of propionyl-CoA synthase from C. aurantiacus indicates that the conversion of 3-hydroxypropionate to propionyl-CoA via the 3-hydroxypropionate route has evolved independently in these two phyla.     

Growth and protection against oxidative stress in young clones and mature spruce trees (Picea abies L.) at high altitudes

Clones of Norway spruce (Picea abies L.) were grown for several years on an altitudinal gradient (1750 m, 1150 m and 800 m above sea level) to study the effects of environmental × genetic interactions on growth and foliar metabolites (protein, pigments, antioxidants). Clones at the tree line showed 4.3-fold lower growth rates and contained 60% less chlorophyll (per gram of dry matter) than those at valley level. The extent of growth reduction was clone-dependent. The mortality of the clones was low and not altitude-dependent. At valley level, but not at high altitude, needles of mature spruce trees showed lower pigment and protein concentrations than clones. In general, antioxidative systems in needles of the mature trees and young clones did not increase with increasing altitude. Needles of all trees at high altitude showed higher concentrations of dehydroascorbate than at lower altitudes, indicating higher oxidative stress. In one clone, previously identified as sensitive to acute ozone doses, this increase was significantly higher and the growth reduction was stronger than in the other genotypes. This clone also displayed a significant reduction in glutathione reductase activity at high altitude. These results suggest that induction of antioxidative systems is apparently not a general prerequisite to cope with altitude in clones whose mother plants originated from higher altitudes (about 650–1100 m above sea level, Hercycnic-Carpathian distribution area), but that the genetic constitution for maintenance of high antioxidative protection is important for stress compensation at the tree line. Received: 13 October 1998 / Accepted: 22 June 1999     

Cytokines promote Wnt signaling and inflammation and impair the normal differentiation and lipid accumulation in 3T3-L1 preadipocytes

Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFalpha impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein alpha, in contrast to peroxisome proliferator-activated receptor gamma, was markedly reduced. TNFalpha also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFalpha maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and beta-catenin levels. TNFalpha, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFalpha prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation.     

Laser scanning cytometry in human brain slices.

BACKGROUND: The Laser Scanning Cytometry (LSC) offers quantitative fluorescence analysis of cell suspensions and tissue sections. METHODS: We adapted this technique to immunohistochemical labelled human brain slices. RESULTS: We were able to identify neurons according to their labelling and to display morphological structures such as the lamination of the entorhinal cortex. Further, we were able to distinguish between neurons with and without cyclin B1 expression and we could assign the expression of cyclin B1 to the cell islands of layer II and the pyramidal neurons of layer V of the entorhinal cortex in Alzheimer's disease effected brain. In addition, we developed a method depicting the three-dimensional distribution of the cells in intact tissue sections. CONCLUSIONS: In this pilot experiments we could demonstrate the power of the LSC for the analysis of human brain sections.     

Solid-state NMR sequential assignments of α-synuclein

Parkinson’s disease is amongst the most frequent and most devastating neurodegenerative diseases. It is tightly associated with the assembly of proteins into high-molecular weight protein species, which propagate between neurons in the central nervous system. The principal protein involved in this process is α-synuclein which is a structural component of the Lewy bodies observed in diseased brain. We here present the solid-state NMR sequential assignments of a new fibrillar form of this protein, the first one with a well-ordered and rigid N-terminal part.     

Exome sequencing identifies a nonsense mutation in <Emphasis Type="Italic">Fam46a</Emphasis> associated with bone abnormalities in a new mouse model for skeletal dysplasia

We performed exome sequencing for mutation discovery of an ENU (N-ethyl-N-nitrosourea)-derived mouse model characterized by significant elevated plasma alkaline phosphatase (ALP) activities in female and male mutant mice, originally named BAP014 (bone screen alkaline phosphatase #14). We identified a novel loss-of-function mutation within the Fam46a (family with sequence similarity 46, member A) gene (NM_001160378.1:c.469G>T, NP_001153850.1:p.Glu157*). Heterozygous mice of this mouse line (renamed Fam46a ^E157*Mhda) had significantly high ALP activities and apparently no other differences in morphology compared to wild-type mice. In contrast, homozygous Fam46a ^E157*Mhda mice showed severe morphological and skeletal abnormalities including short stature along with limb, rib, pelvis, and skull deformities with minimal trabecular bone and reduced cortical bone thickness in long bones. ALP activities of homozygous mutants were almost two-fold higher than in heterozygous mice. Fam46a is weakly expressed in most adult and embryonic tissues with a strong expression in mineralized tissues as calvaria and femur. The FAM46A protein is computationally predicted as a new member of the superfamily of nucleotidyltransferase fold proteins, but little is known about its function. Fam46a ^E157*Mhda mice are the first mouse model for a mutation within the Fam46a gene.