Immunological studies on lysosomal sphingomyelinase: Identification of a 28 000-Da component deficient in urine from patients with Niemann-Pick disease types A and B

The immunoblotting technique was used to identify sphingomyeJinase protein in samples of tissue and urine after subjection to poIyacrylamide-gel etectrophoresis in the presence of sodium dodecyl sulphate. In a sphingomyelinase preparation purified from control urine a prominent band was seen with an M_r of 28 000 Da. Glycoprotein fractions from urine and placenta, a membrane extract from spleen, and a partially purified sphingomyelinase preparation from placenta contained the 28 000-Da band plus additional, higher-M_r bands. The 28 000-Da band was detectable in urine from a patient with Niemann-Pick disease type C, but not in urine from patients with Niemann-Pick disease types A and B. It is concluded t h a t sphingomyeJinase is composed of at least one polypeptide with an M_r of 28 000 Da and that this polypeptide is deficient in the urine of patients with Niemann-Pick disease types A and B.     

The epsilon-subunit of ATP synthase from bovine heart mitochondria. Complementary DNA sequence, expression in bovine tissues and evidence of homologous sequences in man and rat.

The epsilon-subunit of ATP synthase from bovine heart mitochondria is assembled into the extrinsic membrane sector, F1-ATPase. The mature protein is 50 amino acid residues in length and its function is unknown. It is a nuclear gene product that is imported into the organelle. A mixture of 64 oligonucleotides 17 bases long, designed on the basis of the known protein sequence, was synthesized and used as a hybridization probe to isolate a cognate cDNA clone from a bovine library. The DNA sequence of this clone was determined, and the protein sequence of the epsilon-subunit deduced from it agrees exactly with that determined by direct sequence analysis of the protein isolated from bovine hearts. The bovine cDNA was used as a hybridization probe to examine the expression of the epsilon-subunit in various bovine tissues. mRNAs related to the cDNA are found in all of these tissues, and no evidence was obtained of the presence of mRNAs for the epsilon-subunit with similar coding sequences and dissimilar 3' non-coding regions. By hybridization experiments with digests of DNA from cow, man and rat it has been shown that sequences related to the bovine cDNA are present in the genomes of all three species. More than one related sequence was detected in all cases, indicating the presence in all three genomes of more than one gene and/or pseudogenes.     

Branched-chain amino acid aminotransferase in mouse testicular tissue

Branched-chain amino acid aminotransferase (L-leucine:2-oxoglutarate aminotransferase, EC 2.6.1.6) activity was determined in several tissues of the mouse. Testis homogenates presented a specific activity very close to that of heart extracts which were the most active. Enzyme activity was detectable in testes from 5-day-old mice and increased steadily during development to reach a maximum at the 20th day of life. The transaminase was present in the cytosol of testicular homogenates and also associated, probably in the matrix, with a special type of mitochondria present in spermatozoa and gametogenic cells. The enzyme from testis is active against the three branched-chain amino acids and catalyses the reaction in both directions. Highest activity and lowest Km were obtained with L-leucine. Activity with L-valine was the lowest. The enzyme from the mitochondrial fraction showed identical properties to that from the soluble phase. The possible participation of this aminotransferase in a shuttle system transferring reducing equivalents from cytoplasm to mitochondria is postulated.     

Early developmental expression of a normally tumor-associated and drug-inhibited cell surface-located NADH oxidase (ENOX2) in non-cancer cells

Full length mRNA to a drug-inhibited cell surface NADH oxidase, tNOX or ENOX2, is present in both non-cancer and cancer cells but is translated only in cancer cells as alternatively spliced variants. ENOX2 is a growth-related protein of the external plasma membrane surface that is shed into the circulation and is inhibited by a series of quinone site inhibitors with anticancer activity. To test the possibility that ENOX2 expression might be important to early stages of non-cancer cell development, the expression of the protein was monitored in chicken embryos during their development. Polyclonal antisera to a 34 kDa human serum form of ENOX2 cross-immunoreactive with the drug-responsive NADH oxidase of chicken hepatoma cells was used. The protein was identified based on drug-responsive enzymatic activities and analyses by western blots. The drug-responsive activity was associated with plasma membranes and sera of early chicken embryos and with chicken hepatoma plasma membranes but was absent from plasma membranes prepared from livers or from sera of normal adult chickens and from late embryo stages. The findings suggest that ENOX2 may fulfill some functions essential to the growth of early embryos which are lost in late embryo stages and absent from normal adult cells but which then reappear in cancer.     

The removal of extracellular fibronectin from areas of cell-substrate contact

Immunofluorescent labeling for fibronectin was largely excluded from sites of closest contact between spreading chicken gizzard fibroblasts and the substratum. This was observed by double immunofluorescent labeling of fixed cells for fibronectin and vinculin, a smooth muscle intracellular protein that is specifically associated with focal adhesion plaques, in conjunction with interference-reflection microscopy. When the cells were plated on a fibronectin-coated substratum they adhered to its surface and rapidly spread on it. The immunofluorescent labeling for fibronectin in those cultures (after fixation and triton permeabilization) was usually absent from the newly formed, vinculin-containing focal adhesion plaques. We have found, however, that the accessibility to the cell-substrate gap at the focal adhesion plaques is limited and therefore a more direct approach was adopted. We have found that cells spreading on a substrate coated with rhodamine-labeled fibronectin progressively removed the underlying protein from the substrate. The removal of fibronectin involved at least two distinct mechanisms. Part of the substrate-associated fibronectin was removed from small areas and displaced toward the cell center. The arrowhead-shaped areas from which fibronectin was removed often coincided with vinculin-rich focal contacts. We observed, however, many areas where focal contacts were found over unperturbed fibronectin carpet, as well as fibronectin-free areas with no overlapping focal contacts. The possibilities that fibronectin is actively displaced from areas of cell-substrate contact, that the focal adhesion plaques are transiently associated with these areas and their implications on the dynamics of cell spreading and locomotion are discussed. The second route of fibronectin removal from the substrate was endocytosis. The rhodamine-labeled fibronectin was found in the cells in a partial or transient association with clathrin-containing structures.     

THE DETERMINATION OF ACETYLCHOLINESTERASE ACTIVITY OF BRAIN SLICES AND ITS SIGNIFICANCE IN STUDIES OF EXTRACELLULAR ACETYLCHOLINESTERASE

The AChE activity of single slices obtained from the surface of the temporoparietal region of rat brains was measured colorimetrically under anaerobic conditions with acetylthiocholine as substrate. In intact slices from untreated rats AChE activity was only a small proportion of that of homogenates made from the slices, but this proportion increased with the surface area to weight ratio of slices and with an increase in substrate concentration, intact slices not showing substrate inhibition. The inhibition of AChE determined in slices from rats treated with DFP or paraoxon was less than that in homogenates obtained from the slices. When the access of substrate was limited to the cut surface of a slice, the rate of hydrolysis was four times greater than that observed when access was solely from the uncut surface. It is concluded that under anaerobic conditions the substrate diffuses into slices to a depth which is not constant but a function of both substrate and enzyme concentration. Thus the AChE activity of slices cannot be used as a measure of extracellular AChE.     

Purification and quantitation of a rat plasma selenoprotein distinct from glutathione peroxidase using monoclonal antibodies

Studies with 75Se have shown the existence of a rat plasma selenoprotein in addition to glutathione peroxidase. Because the function of the protein is not known, it has been referred to as selenoprotein P. A partially purified preparation was used to produce a monoclonal antibody to selenoprotein P. The antibody did not bind glutathione peroxidase as evidenced by its failure to remove glutathione peroxidase activity from rat plasma by immunoprecipitation. An immunoaffinity column was prepared with the monoclonal antibody, and selenoprotein P was purified 1270-fold from rat plasma in a two-step procedure. The purified selenoprotein P migrated in a single band with an Mr of 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography demonstrated that this band contained 75Se when the protein was purified from rats which had received 75SeO2-(3). A competitive radioimmunoassay for selenoprotein P was developed. The selenoprotein P concentration in plasma of selenium-replete rats was determined with this assay to be 51 +/- 3.7 micrograms/ml. It was less than 5 micrograms/ml in plasma from selenium-deficient rats. Injection of 50 micrograms of selenium into selenium-deficient rats caused an increase in selenoprotein P from less than 10% of control to 52% of control in 6 h. Plasma glutathione peroxidase activity increased only from 2.2 to 3.1% of control. These experiments demonstrate that rat plasma contains a selenoprotein distinct from glutathione peroxidase. The concentration of this selenoprotein is depressed in selenium deficiency, as is glutathione peroxidase activity, but selenoprotein P increases more rapidly when selenium is supplied than does glutathione peroxidase activity.     

Role of mannosyl lipid intermediate in the synthesis of Neurospora crassa glycoproteins.

Particulate membrane preparations from Neurospora crassa incorporated mannose from GDP-[14C] mannose into endogenous lipid and particulate protein acceptors. Synthesis of the mannosyl lipid is reversible in the presence of GDP. Chemical and chromatographic characterization of the mannosyl lipid suggest that it is a mannosylphosphorylpolyisoprenol. The other endogenous acceptor was precipitated by trichloracetic acid. Gel filtration and electrophoresis studies before and after treatment with proteolytic enzymes indicate that the second acceptor is a glycoprotein(s). beta Elimination studies on the mannosyl protein formed from GDP-[14C] mannose with Mg2+ in the reaction mixture or formed from mannosyl lipid indicate thad with the peptide chain. Several lines of evidence indicate that in Neurospora crassa the mannosyl lipid is an obligatory intermediate in the in vitro mannosylation of the protein. (a) At 15 degrees C the initial formation of the mannosyl lipid is faster than the initial formation of the mannosyl protein. (b) Exogenous partially purified mannosyl lipid can function as a mannosyl donor for the synthesis of the mannosyl protein. This reaction was also dependent on a divalent metal. The rate of this reaction was optimal at a concentration of Triton X-100 which effectively inhibited the transfer of mannose from GDP-[14C] mannose to lipid and protein, indicating that GDP-mannose was not an intermediate in the transfer of mannose from lipid to protein. The mannosyl protein formed in this reaction was indistinguishable by several criteria from the mannosyl protein formed from GDP-[14C] mannose and Mg2+. (c) The effect of a chase with an excess of unlabeled GDP-mannose on the incorporation of mannose into endogenous acceptors was immediate cessation of the synthesis and subsequent turnover of the mannosyl lipid; in contrast, however, incorporation of mannose into protein continued and was proportional to the loss of mannose from the mannosyl lipid.     

CFD transient simulation of the cough clearance process using an Eulerian wall film model

In this study, a cough cycle is reproduced using a computational methodology. The Eulerian wall film approach is proposed to simulate airway mucus flow during a cough. The reproduced airway domain is based on realistic geometry from the literature and captures the deformation of flexible tissue. To quantify the overall performance of this complex phenomenon, cough efficiency (CE) was calculated, which provided an easily reproducible measurement parameter for the cough clearance process. Moreover, the effect of mucus layer thickness was examined. The relationship between the CE and the mucus viscosity was quantified using reductions from 20 to 80%. Finally, predictions of CE values based on healthy person inputs were compared with values obtained from patients with different respiratory diseases, including chronic obstructive pulmonary disease (COPD) and respiratory muscle weakness (RMW). It was observed that CE was reduced by 50% in patients with COPD compared with that of a healthy person. On average, CE was reduced in patients with RMW to 10% of the average value of a healthy person.     

Interaction of adenylosuccinate synthetase with F-actin

Both crude and purified preparations of adenylosuccinate synthetase from muscle were found to combine with, and dissociate from, muscle debris precipitated from a homogenate of the muscle with water. The binding and dissociation depended on ionic strength. Further study showed that the muscle enzyme was adsorbed to F-actin, but not to G-actin or myosin. The muscle-type enzyme from the liver also associated with F-actin, but the liver-type enzyme from the liver did not. In the absence of KCl the molar ratio of adenylosuccinate synthetase from skeletal muscle to actin monomer in F-actin in the complex formed was 1 to 4. From a Scatchard plot the dissociation constant was calculated to be 0.72 micrometer. The binding was maximal at pH 5.5-7 in 30 mM potassium phosphate buffer. The complex was completely dissociated in the presence of 0.21 M KCl. The physiological significance of this binding is discussed on the basis of these findings.     

Characterization of a spirochaete isolated from a case of bovine digital dermatitis

AIMS: The aim of the study was to characterize a spirochaete isolated from the lesions of a cow with digital dermatitis (DD). METHODS AND RESULTS: The characterization was on the basis of its light and electron microscopic appearance, enzymic profile and DNA sequence analysis of its flagellin and 16S rRNA genes. The spirochaete was 6-8-microm long and 0.2-0.3 microm in diameter, and possessed seven to eight periplasmic flagella, with three to five helical turns. The enzymic profile of the bacterium resembles, but is not identical to that of Treponema brennaborense. Its flagellin gene sequence was identical to that of Treponema phagedenis but distinct from that of an ovine spirochaete. Analysis of a 1477-bp region of the 16S rRNA genes indicated that this is a Treponema species and that it is indistinguishable from some isolates made from cases of bovine DD in the United States. Finally, electron microscopy revealed the presence of myovirus-like bacteriophage particles in all cultures of the treponeme examined. CONCLUSIONS: The spirochaete isolate was identified as a Treponema species closely related to some isolates from the United States (by 16S rDNA) and to T. phagedenis (by flagellin gene sequence) and is associated with bacteriophage particles. SIGNIFICANCE AND IMPACT OF THE STUDY: The fact that the isolates with the same or very similar 16S rDNA sequences have been obtained from cases of bovine DD in cattle in different countries at different times, lends further support to the hypothesis that treponemes play a role in the pathogenesis of this disease.     

Resolution and reconstitution of a bound Fe-S protein from the photosynthetic reaction center of Heliobacterium modesticaldum

The photosynthetic reaction center of Heliobacterium modesticaldum (HbRC) was isolated from membranes using n-dodecyl beta-D-maltopyranoside followed by sucrose density ultracentrifugation. The low-temperature EPR spectra of whole cells, isolated membranes, and HbRC complexes are similar, showing a single Fe-S cluster with g values of 2.067, 1.933, and 1.890 after illumination at 20 K, and a complex spectrum attributed to exchange interaction from two Fe-S clusters after illumination during freezing. The protein containing the Fe-S clusters was removed from the HbRC by washing it with 1.0 M NaCl and purified by ultrafiltration over a 30 kDa cutoff membrane. Analysis of the filtrate by SDS-PAGE showed a major band at approximately 8 kDa that was weakly stained with Coomassie Brilliant Blue and strongly stained with silver. The optical spectrum of the oxidized Fe-S protein shows a maximum at 410 nm, and the EPR spectrum of the reduced Fe-S protein shows a complex set of resonances similar to those found in 2[4Fe-4S] ferredoxins. The HbRC core was purified by DEAE ion-exchange chromatography and resolved by SDS-PAGE. The purified HbRC was composed of a band at ca. 40 kDa, which is identified as PshA, and several additional proteins. The isolated Fe-S protein rebinds spontaneously to purified HbRC cores, and the light-induced EPR signals of the Fe-S clusters are recovered. The flash-induced kinetics of the HbRC complex show two kinetic phases at room temperature, one with a lifetime of 75 ms and the other with a lifetime of 15 ms. The 75 ms component is lost when the Fe-S protein is removed from the HbRC complex, and it is regained when the Fe-S protein is rebound to HbRC cores. Thus, the 75 ms kinetic phase is derived from recombination of a terminal Fe-S cluster with P798(+), and the 15 ms kinetic phase is derived from recombination with an earlier acceptor, probably F(X). We suggest that the bound Fe-S protein present in the HbRC be designated PshB.     

An atypical heme-binding structure of cytochrome c1 of Euglena gracilis mitochondrial complex III

Complex III was purified from submitochondrial particles prepared from Euglena gracilis. The purified complex consisted of 10 subunits and lost antimycin sensitivity. The Euglena complex III showed an atypical difference absorption spectrum for cytochrome c1 with its alpha-band maximum at 561 nm. The pyridine ferrohemochrome prepared from covalently bound heme in the Euglena complex III had an alpha-peak at 553 nm. This wavelength is the same as that of pyridine ferrohemochrome prepared from Euglena mitochondrial cytochrome c (c-558), the heme of which is linked to only a single cysteine residue through a thioether bond. Cytochrome c1 which was a heme-stained subunit with a molecular mass of 32.5 kDa was isolated from the purified complex III and its N-terminal sequence of 46 amino acids was determined. On the basis of apparent homologies to cytochromes c1 from other sources, this sequence included the heme-binding region. However, the amino acid at position 36, corresponding to the first cysteine involved in heme linkage in other cytochromes c1, was phenylalanine. Position 39, corresponding to the second cysteine, was not identified despite the treatment for removal of the heme and carboxymethylation of the expected cysteine. The unidentified amino acid is assumed to be a derivative of cysteine to which the heme is linked through a single thioether bond. The histidine-40 corresponding to the probable fifth ligand for heme iron was conserved in Euglena cytochrome c1.     

Clinical muscular sarcocystosis in a dog

Muscular sarcocystosis is a rare infection in dogs. Clinical myositis associated with an unidentified species of Sarcocystis was diagnosed in an adult dog from Canada. There was granulomatous myositis associated with numerous immature sarcocysts in a muscle biopsy obtained from the dog. The sarcocysts were up to 550 microm long and up to 45 microm wide. The sarcocyst wall was approximately 1 microm thick and contained short, stubby, villar protrusions that lacked microtubules. This is the first report on clinical muscular sarcocystosis in a dog.     

Small-angle X-ray-scattering investigation and structural-model study of the fatty-acid synthetase from pig liver (author's transl)

The structure of the fatty acid synthetase from pig liver was studied on models based upon structural and functional properties selected from pertinent results available from numerous investigations carried out with fatty acid synthetases from this and other sources. When comparing small-angle X-ray-scattering curves calculated with these models and curves obtained from small-angle X-ray-scattering experiments carried out with the pig-liver enzyme, we tried to select a model which would lead to an acceptable correlation between the calculated and the experimental curves and at the same time fulfil the known structural and functional requirements. The comparison of the curves was started with a model of low complexity. The observed discrepancy, together with arguments from the structural and the functional properties, helped decide which is the next most reasonable model to be considered. This procedure was repeated for five models of increasing complexity. In the model which led to the best fit the multienzyme complex is composed of two halves in an assymetric conformation including hollow spaces. This highly anisotropic model would imply that the two halves change their conformation each time a synthetic cycle is completed and that the growing fatty acid is handed over from one half to the other.     

Activation of the hole-forming toxin aerolysin by extracellular processing.

A precursor-product relationship between aerolysin and a protein with a higher molecular weight was observed in culture supernatants of Aeromonas hydrophila. The larger protein was isolated by ammonium sulfate precipitation and ion-exchange and hydroxyapatite chromatography and compared with purified aerolysin. It was at least 250 times less hemolytic than aerolysin. Both proteins had the same amino acid sequence at the amino terminus. Cyanogen bromide fragments obtained from the two were identical except that each protein contained one unique fragment, and the fragment from the larger protein was 2,500 daltons larger than the fragment obtained from aerolysin. Treatment with trypsin or with an extracellular Aeromonas protease resulted in rapid conversion of the larger protein to a form corresponding in molecular weight and activity to aerolysin. The results indicate that aerolysin is exported to the culture supernatant as a protoxin which is later activated by proteolytic removal of a peptide from the C terminus.     

Pellet pestle homogenization of agarose gel slices at 45 degrees C for deoxyribonucleic acid extraction

A simple method for extracting DNA from agarose gel slices is described. The extraction is rapid and does not involve harsh chemicals or sophisticated equipment. The method involves homogenization of the excised gel slice (in Tris-EDTA buffer), containing the DNA fragment of interest, at 45 degrees C in a microcentrifuge tube with a Kontes pellet pestle for 1 min. The "homogenate" is then centrifuged for 30 s and the supernatant is saved. The "homogenized" agarose is extracted one more time and the supernatant obtained is combined with the previous supernatant. The DNA extracted using this method lent itself to restriction enzyme analysis, ligation, transformation, and expression of functional protein in bacteria. This method was found to be applicable with 0.8, 1.0, and 2.0% agarose gels. DNA fragments varying from 23 to 0.4 kb were extracted using this procedure and a yield ranging from 40 to 90% was obtained. The yield was higher for fragments 2.0 kb and higher (70-90%). This range of efficiency was maintained when the starting material was kept between 10 and 300 ng. The heat step was found to be critical since homogenization at room temperature failed to yield any DNA. Extracting DNA with our method elicited an increased yield (up to twofold) compared with that extracted with a commercial kit. Also, the number of transformants obtained using the DNA extracted with our method was at least twice that obtained using the DNA extracted with the commercial kit.     

Glycogen synthesis in the obliquely striated muscle of Ascaris suum

A glycogen synthase, designated GS II, which occurs in a protein/carbohydrate complex has been purified from Ascaris suum muscle. The purified GS-II complex which is eluted from concanavalin-A--Sepharose contains proteins with Mr 140,000 and 66,000 and a glycoprotein with a carbohydrate/protein mass ratio of 3:1. GS II activity was totally dependent on glucose 6-phosphate, but exogenous glycogen was not required for polysaccharide synthesis. The GS-II complex was not phosphorylated by cyclic-AMP-dependent protein kinase, and antibodies to the protein and carbohydrate components of GS II did not cross react with the purified cyclic-AMP-regulated glycogen synthase (GS I) from A. suum muscle. Polysaccharide which was synthesized de novo by the complex was added to the large-molecular-mass glycoprotein in GS II. The glycogen-like character of the newly synthesized polysaccharide was confirmed by the observation that glycogen phosphorylase utilized the polymer as substrate in both the synthesis and degradation reactions. A model is discussed in which a core glycoprotein serves as the substrate for a glycogen synthase which is distinctly different from GS I.     

Cladistic analysis and osteological descriptions of the frog species in the Leptodactylus fuscus species group (Anura, Leptodactylidae)

The genus Leptodactylus is predominantly Neotropical (a few species have colonized the southern Neartic region) and is distributed from Texas to Argentina and on certain Caribbean islands. Leptodactylus was divided into five groups of species: Leptodactylus melanonotus , Leptodactylus ocellatus, Leptodactylus fuscus , Leptodactylus pentadactylus and Leptodactylus marmoratus . Among these, the L. fuscus group is the one with most species, with 27 taxa. Characters unverified in most of the species are used to define the L. fuscus group. However, the monophyly of the group has never been tested rigorously in a quantitative phylogenetic context. Thus, the main goal of this study was to test such monophyly and to construct a phylogeny of the L. fuscus group. A matrix of 114 characters scored across 43 taxa was constructed, with 31 characters taken from external morphology, 58 from adult skeletons, 16 from larval chondrocranium, 5 from ethology and 4 from morphometric data were included. Out of all the species examined, 23 belonged to the ingroup and 20 to the outgroup. The data set was analysed with implied weights, by using TNT software. The monophyly of the group was strongly supported in the fittest cladogram obtained. The optimizations of some characters on this hypothesis support traditional evolutionary hypotheses. The optimizations also suggest the presence of paedomorphic character states in some species, which is also discussed.