The Ighmbp2 helicase structure reveals the molecular basis for disease-causing mutations in DMSA1

Mutations in immunoglobulin µ-binding protein 2 (Ighmbp2) cause distal spinal muscular atrophy type 1 (DSMA1), an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. However, despite extensive studies, the mechanism of disease-causing mutations remains elusive. Here we report the crystal structures of the Ighmbp2 helicase core with and without bound RNA. The structures show that the overall fold of Ighmbp2 is very similar to that of Upf1, a key helicase involved in nonsense-mediated mRNA decay. Similar to Upf1, domains 1B and 1C of Ighmbp2 undergo large conformational changes in response to RNA binding, rotating 30° and 10°, respectively. The RNA binding and ATPase activities of Ighmbp2 are further enhanced by the R3H domain, located just downstream of the helicase core. Mapping of the pathogenic mutations of DSMA1 onto the helicase core structure provides a molecular basis for understanding the disease-causing consequences of Ighmbp2 mutations.     

Temporal relationship between nerve-stump-length-dependent changes in the autophosphorylation of a cyclic AMP-dependent protein kinase and the acetylcholine receptor content in skeletal muscle

The acetylcholine receptor (AChR) content and the autorphosphorylation of the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) were evaluated in rat soleus muscles at 24, 30 and 66 hr after surgical denervation by cutting the nerve at a short distance (short-nerve-stump) and at a long distance (long-nerve-stump) from the muscle. AChR content was based on the specific binding of [¹²⁵I]alpha-bungarotoxin (BUTX); changes in the autophosphorylation of R-II were based upon the predominant in vitro³²P-phosphorylation of a 56-Kd soluble protein in cytosolic fractions of solei. The AChR content and the³²P-autophosphorylation of R-II were increased in samples from short-nerve-stump solei, but not from long-nerve-stump solei, after a denervation-time of 30 hr. This nerve-stump-length dependency indicates that the two denervation effects are not related to the immediate halt of impulse-evoked muscle contractility. Furthermore, the results show that alterations in the³²P-autophosphorylation of R-II occurred before, as well as whenever, increases in the AChR content were found. Speculatively, this temporal relationship may be significant with respect to the potential role of R-II in gene expression.Abbreviations ACh acetylcholine - AChR acetylcholine receptor(s) - BUTX alpha-bungarotoxin - Kd kilodalton - PAGE polyacrylamide gel electrophoresis - R-II regulatory subunit of cyclic AMP-dependent protein kinase type II - SDS sodium dodecyl sulfate     

Proteinases and the instability of isocitrate lyase in extracts of developing flax seedlings

In extracts of flax seedlings 4 days after imbibition, isocitrate lyase activity is unstable in comparison to that in extracts from 2.5-day seedlings or to malate syntheses analysed at several stages of development. This instability in extracts of 4-day seedlings is especially pronounced when a large number of seedlings is homogenized per unit volume of Tris-Mg²⁺-EDTA-dithioerythritol buffer. However, isocitrate lyase can be stabilized when the resultant homogenate is diluted soon after seedling breakage. The pronounced instability in more concentrated extracts is not due to inadequate buffering by the homogenization medium, nor can it be due to polyphenols because added polyvinylpyrrolidone has no effect. Mixing of a heated supernatant from concentrated extract with dilute unheated extract yields the units of stable isocitrate lyase expected in the dilute extract, ruling out stoichiometric inactivation by a heat-stable component. The pronounced instability is attributed to the action of proteinases. A theoretical model assuming a decay process that is first order in isocitrate lyase and first-order in one or more proteinases is in reasonable agreement with the results. Malate synthase and NADP⁺-isocitrate dehydrogenase are much more stable in concentrated extracts prepared from 4-day flax seedlings. Isocitrate lyase is stable in concentrated extracts of 5-day watermelon seedlings, which is a developmental stage analogous to that for 4-day flax seedlings.     

Nachweis der Phytochrom-induzierten de novo-Synthese von Phenylalaninammoniumlyase (PAL,E.C.4.3.1.5) in Keimlingen von Sinapis alba L. durch Dichtemarkierung mit Deuterium

Summary Using the in vivo density labeling technique with deuterium oxide it is confirmed that during phytochrome mediated photomorphogenesis in mustard seedlings a true de novo synthesis of phenylalanine ammonia-lyase is induced by active phytochrome (P _fr).     

Fluorescence immunolocalization of bound atrazine residues in plant tissue

Bound atrazine was detected inElodea canadensis by an improved immunohistochemical fluorescence procedure using anti-triazine antibodies from rabbits, biotin-labelled anti-rabbit immunoglobulin G and streptavidin-phycoerythrin conjugate. Whereas no labelling was found in control plants grown in charcoal-filtered, atrazine-free water, the labelling of plants obtained from their natural habitat and grown in tap water was sometimes nearly as high as in samples loaded with atrazine. The efficiency of the immunofluorescence procedure was compared using several antisera obtained by immunizing with different hapten conjugates and purified by various purification methods. The best results were observed with the atrazine analogue ametryn sulfoxide, which was coupled to bovine serum albumin for immunization and to Sepharose for immunoaffinity chromatography. The procedure described in this paper may serve as a general tool for detecting bound pesticide residues in plant material. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Nutrient uptake relationship to root characteristics of rice

Data on root parameters and distribution are important for an improved understanding of the factors influencing nutrient uptake by a crop. Therefore, a study was conducted on a Crowley silt loam at the Rice Research and Extension Center near Stuttgart, Arkansas to measure root growth and N, P and K uptake by three rice (Oryza sativa L.) cultivars at active tillering (36 days after emergence (DAE)), maximum tillering (41 DAE), 1.25 cm internode elongation (55 DAE), booting (77 DAE) and heading (88 DAE). Soil-root core samples were taken to a depth of 40 cm after plant samples were removed, sectioned into 5 cm intervals, roots were washed from soil and root lengths, dry weights and radii were measured. Root parameters were significantly affected by the soil depth × growth stage interaction. In addition, only root radius was affected by cultivar. At the 0- to 5-cm soil depth, root length density ranged from 38 to 93 cm cm^-3 throughout the growing season and decreased with depth to about 2 cm cm^-3 in the 35- to 40-cm depth increment. The increase in root length measured with each succeeding growth stage in each soil horizon also resulted in increased root surface area, hence providing more exposed area for nutrient uptake. About 90% of the total root length was found in the 0- to 20-cm soil depth throughout the season. Average root radius measured in the 0- to 5-cm and 35- to 40-cm depth increments ranged from 0.012 to 0.013 cm and 0.004 to 0.005 cm, respectively throughout the season. Total nutrient uptake by rice differed among cultivars only during vegetative growth. Differences in total nutrient uptake among the cultivars in the field appear to be related to absorption kinetics of the cultivars measured in a growth chamber study. Published with permission of the Arkansas Agricultural Experiment Station.     

The Mammalian Protein (rbet1) Homologous to Yeast Bet1p Is Primarily Associated with the Pre-Golgi Intermediate Compartment and Is Involved in Vesicular Transport from the Endoplasmic Reticulum to the Golgi Apparatus

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively