TDP-1, a toxic component causing tibial dyschondroplasia in broiler chickens, and trichothecenes from Fusarium roseum 'Graminearum'.

Fusarium roseum 'Graminearum' was isolated from overwintered oats in Alaska and was tested for its ability to cause tibial dyschondroplasia (TDP) in broiler chickens. The water-soluble fraction was tested and found to cause TDP. In addition, diacetoxyscirpenol and 7-hydroxydiacetoxyscirpenol were identified in the acetonitrile fraction of the extracts and caused mild mouth lesions in chickens. Six major water-soluble components were purified by thin-layer chromatography and tested for toxicity to chick embryos. One of the six components, called TDP-1, was found to be lethal to chick embryos. There was a 100% incidence of TDP in chickens fed a diet containing 75 ppm (wt/wt) of pure TDP-1, thus establishing the cause and effect relationship between TDP and TDP-1. Analyses by thin-layer chromatography and mass spectrometry revealed that TDP-1 is polar and ninhydrin positive, exhibits fluorescence with UV irradiation, and is a nitrogen-containing component with an empirical formula of C15H20N2O4.     

Modulation of assembly of TDP-43 low-complexity domain by heparin: From droplets to amyloid fibrils

TAR DNA-binding protein 43 (TDP-43) is an RNA-regulating protein that carries out many cellular functions through liquid-liquid phase separation (LLPS). The LLPS of TDP-43 is mediated by its C-terminal low-complexity domain (TDP43-LCD) corresponding to the region 267–414. In neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia, pathological inclusions of the TDP-43 are found that are rich in the C-terminal fragments of ～25 and ～35 kDa, of which TDP43-LCD is a part. Thus, understanding the assembly process of TDP43-LCD is essential, given its involvement in the formation of both functional liquid-like assemblies and solid- or gel-like pathological aggregates. Here, we show that the solution pH and salt modulate TDP43-LCD LLPS. A gradual reduction in the pH below its isoelectric point of 9.8 results in a monotonic decrease of TDP43-LCD LLPS due to charge-charge repulsion between monomers, while at pH 6 and below no LLPS was observed. The addition of heparin to TDP43-LCD solution at pH 6, at a 1:2 heparin-to-TDP43-LCD molar ratio, promotes TDP43-LCD LLPS, while at higher concentration, it disrupts LLPS through a reentrant phase transition. Upon incubation at pH 6, TDP43-LCD undergoes gelation without phase separation. However, in the reentrant regime in the presence of a high heparin concentration, it forms thick amyloid aggregates that are significantly more SDS resistant than the gel. The results indicate that the material nature of the TDP43-LCD assembly products can be modulated by heparin which is significant in the context of liquid-to-solid phase transition observed in TDP-43 proteinopathies. Our findings are also crucial in relation to similar transitions that could occur due to alteration in the molecular level interactions among various multivalent biomolecules involving other LCDs and RNAs.     

Composition of Acid-sensitive Fraction in Soybean Proteins

An acid-sensitive fraction (ASF) was prepared from defatted soybean meals by two procedures. ASF1 was prepared by precipitation at pH 4.5 followed by removal of 1 m NaCl-soluble materials from the precipitate. ASF2 was prepared by precipitation in solution containing 1 m NaCl at pH 4.5. The protein components of the two fractions were analyzed by gel electrophoresis in a dissociating-buffer system and found to contain β-conglycinin, glycinin and whey proteins. In addition to these, several other bands appeared.

Appreciable amounts of lipid (8.2% in ASF1 and 8.8% in ASF2) were also found in the fractions. They were separated by column chromatography and thin-layer chromatography. Glycolipids were the major components of the lipids. Both glycolipid and phospholipid fractions contained slower-moving materials on thin-layer chromatography.     

The effect of antioxidant-deficiency on phospholipid composition in the chick brain

Abstract— A significant increase in arachidonate was noted in the total phospholipids of brain of chicks with nutritional antioxidant-deficiency and encephalomalacia. After thin-layer chromatography of the brain lipids, this increase in arachidonate was found to be restricted to the phosphatidyl serine fraction. Significant decreases in docosahexaenoate and docosapentaenoate were noted in the phosphatidyl ethanolamine fraction. The changes in fatty acid composition of phospholipids in chick brain are comparable to those previously observed in phospholipids of skeletal muscle, liver and testes of the rat.     

Coaggregation of RNA-binding proteins in a model of TDP-43 proteinopathy with selective RGG motif methylation and a role for RRM1 ubiquitination

TAR DNA-binding protein 43 (TDP-43) is a major component within ubiquitin-positive inclusions of a number of neurodegenerative diseases that increasingly are considered as TDP-43 proteinopathies. Identities of other inclusion proteins associated with TDP-43 aggregation remain poorly defined. In this study, we identify and quantitate 35 co-aggregating proteins in the detergent-resistant fraction of HEK-293 cells in which TDP-43 or a particularly aggregate prone variant, TDP-S6, were enriched following overexpression, using stable isotope-labeled (SILAC) internal standards and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We also searched for differential post-translational modification (PTM) sites of ubiquitination. Four sites of ubiquitin conjugation to TDP-43 or TDP-S6 were confirmed by dialkylated GST-TDP-43 external reference peptides, occurring on or near RNA binding motif (RRM) 1. RRM-containing proteins co-enriched in cytoplasmic granular structures in HEK-293 cells and primary motor neurons with insoluble TDP-S6, including cytoplasmic stress granule associated proteins G3BP, PABPC1, and eIF4A1. Proteomic evidence for TDP-43 co-aggregation with paraspeckle markers RBM14, PSF and NonO was also validated by western blot and by immunocytochemistry in HEK-293 cells. An increase in peptides from methylated arginine-glycine-glycine (RGG) RNA-binding motifs of FUS/TLS and hnRNPs was found in the detergent-insoluble fraction of TDP-overexpressing cells. Finally, TDP-43 and TDP-S6 detergent-insoluble species were reduced by mutagenesis of the identified ubiquitination sites, even following oxidative or proteolytic stress. Together, these findings define some of the aggregation partners of TDP-43, and suggest that TDP-43 ubiquitination influences TDP-43 oligomerization.     

Water soluble products of UV-irradiated autoxidized linoleic and linolenic acids

The water-soluble products of the UV-initiated autoxidation of linoleic and linolenic acids emulsified in water were separated into volatile and relatively involatile components, each of which reacted with both thiobarbituric acid (TBA) and peroxidase. The volatile TBA-reactive compound is probably malonaldehyde and the volatile peroxidase-reactive compound is hydrogen peroxide. Additional compounds which absorb UV light were present in the volatile fraction. After thin-layer chromatography of the involatile fraction, reactivity toward TBA and peroxidase was found in the same spot. Approximate molar yields of hydrogen peroxide, malonaldehyde, "hydroperoxides", and other TBA-reactive compounds were estimated. The ratio of "hydroperoxide" to TBA reactivity was lower for linoleic than for linolenic acid. The mass of relatively involatile compounds was about 20 times greater than that predicted from either peroxidase or TBA assays of water extracts of oxidized linolenic acid. The properties of the water extract were similar to those shown by others for the products of prolonged autoxidation (without UV-irradiation) of emulsified methyl linoleate.     

Purification and cDNA cloning of a protein derived from Flammulina velutipes that increases the permeability of the intestinal Caco-2 cell monolayer.

A new protein that decreases transepithelial electrical resistance (TEER) in the human intestinal Caco-2 cell monolayer was found in a water-soluble fraction of the mushroom Flammulina velutipes. This protein, termed TEER-decreasing protein (TDP), is not cytotoxic and does not induce cell detachment, but rapidly increases the tight junctional permeability for water-soluble marker substances such as Lucifer Yellow CH (Mr 457) through the paracellular pathway. TDP was isolated and purified from the aqueous extract of F. velutipes by chromatographic means. Purified TDP was found to be a simple, nonglycosylated protein without intermolecular disulfide bonds, and the apparent molecular mass as estimated by SDS/PAGE and gel filtration is 30 kDa. It was revealed that the N-terminal amino-acid sequence of purified TDP is identical to the recently reported N-terminal sequence of flammutoxin, a membrane-perturbing hemolytic protein, for which the complete primary structure has not yet been reported [Tomita, T., Ishikawa, D., Noguchi, T., Katayama, E., and Hashimoto, Y. (1998) Biochem. J. 333, 24794-24799]. The cDNA coding for TDP was cloned by 5' and 3' rapid amplification of cDNA ends. The ORF encodes a protein with 272 amino-acid residues showing no homology to known proteins. Relevant studies using TDP cDNA will provide insight into the structure-function relationships of membrane pore-forming toxins.     

Synergistic actions of a thrombin-derived synthetic peptide and a thrombin receptor-activating peptide in stimulating fibroblast mitogenesis

We measured the ability of the thrombin receptor activating peptide, SFLLR-NH₂ (P5A) to stimulate ³H-thymidine incorporation in hamster CCL-39 fibroblasts either alone or in combination with the thrombin-derived polypeptides, YPPWNKNFTENDLL (TDP-1) and AGYKPDEGKRGDACEGDSGGPFV (TDP-2). In the presence (but not absence) of the amino peptidase inhibitor amastatin (10 μM), P5A alone (7.5 to 100 μM) caused a 1.5- to 2-fold stimulation of thymidine incorporation above basal, even though this inhibitor did not abrogate the degradation of P5A by other peptidases present in the assay medium. Neither TDP-1 nor TDP-2 alone had any effect on thymidine incorporation. However, TDP-1 (30 to 90 μM) considerably augmented P5A-mediated thymidine incorporation at low P5A concentrations (7.5 to 30 μM), shifting the P5A concentration-effect curve to the left. TDP-2 was inactive in this regard. The EC₅₀ for this potentiating action of TDP-1 was approximately 40 μM. Further, thrombin, rendered proteolytically inactive by a low-molecular-weight bifunctional inhibitor, hirutonin-6, also acted synergistically with P5A to stimulate CCL-39 cell thymidine incorporation. We hypothesize that thrombin can cause its cellular effects, such as thymidine incorporation, not only via the proteolytic activation of its G-protein-coupled receptor, but also via the concurrent and synergistic interaction of its TDP-1 peptide domain with a separate cell surface docking site. © 1996 Wiley-Liss, Inc.     

Dihydroxy and monohydroxy fatty acids in Legionella pneumophila

Five strains of Legionella pneumophila were examined for the presence of hydroxy fatty acid. The cellular distribution of the fatty acids was also determined, as was the variation of hydroxy acid production on five growth media. The strains tested all produced approximately 5 mol% of hydroxy fatty acid, most of which was found in the nonextractable, alkali-stable, acid-labile (wall-associated, amide-linked) fraction. Three major hydroxy acids were found, along with several minor components. The major hydroxy acids were analyzed by thin-layer chromatography, gas-liquid chromatography, mass spectrometry, and infrared spectrophotometry. These compounds were tentatively identified as 3-hydroxy-12-methyltridecanoate, 3-hydroxy-n-eicosanoate, and a novel dihydroxy acid, 2,3-dihydroxy-12-methyltridecanoate. The total amount of hydroxy acid produced, as well as the profile of the hydroxy acids, remained relatively unchanged with respect to strain and growth medium.     

Studies on the Lipid Components of Endotoxin II. Chemical Analyses and Biological Properties of Neutral,Polar-I and Polar-II Subfractions

Lipid components obtained from Salmonella typhosa O-901 endotoxin by acid hydrolysis were separated into neutral, polar-I and polar-II lipid fractions by silica gel column chromatography. These lipids were further separated by silica gel column and/or thin-layer chromatography. The subfractions were analyzed by thin-layer chromatography, gas chromatography and infrared spectrophotometry. Seven subfractions obtained from the neutral lipid fraction contained lauric, myristic, palmitic, 3-OH-myristic acid, artificial products of 3-OH-myristic acid, or a small amount of two unidentified fatty acids. These fatty acids and glucosamine were commonly detected in six subfractions obtained from the polar-I lipid fraction. Fatty acids, glucosamine, and O-phosphorylethanolamine were detected in all of the 13 subfractions obtained from the polar-II lipid fraction. Chick embryo lethal activity, rabbit pyrogenicity and in vitro interferon inducing activity were found in three polar-I lipid subfractions and five polar-II lipid subfractions, but not in neutral lipids. The activities were highest in a polar-II lipid subfraction, which contained smaller amounts of O-phosphorylethanolamine and glucosamine than the other subfractions. However, no particular chemical constituent (s) related to the biological activities could be found. Prolonged acid hydrolysis of the polar-II lipids gave rise to neutral and polar-I lipids. Chemical and biological aspects of the lipid constituents of endotoxin are discussed.     

Immunochemical Analysis of Serologically Active Lipids of Mycoplasma pneumoniae

The major complement-fixing antigen of Mycoplasma pneumoniae is found in the lipid fraction of the organism. When the lipids of M. pneumoniae were fractionated by column chromatography on silicic acid, serological activity against both rabbit and human immune sera was found in two fractions, B and D. Fraction B, eluted with chloroform-methanol (9:1), was a minor component in terms of total complement-fixing activity and contained a complex of lipids which were detected in the region characteristic of phosphatidic acids by thin-layer chromatography on Silica Gel G. Fraction D, eluted with ethyl acetate-methanol (3.5:2), had approximately the same complement-fixing antigen titer as the original lipid extract and appeared as a "comet-shaped" spot between phosphatidylethanolamine and phosphatidylcholine on Silica Gel G plates charred with sulfuric acid. However, by thin-layer chromatography on Silica Gel H impregnated with sodium tetraborate, it was demonstrated that fraction D did contain multiple components, all but one of which were carbohydrate-containing lipids (giving positive reactions when sprayed with orcinol-sulfuric acid reagent). Fraction D was found to contain glycerol and phosphate in equimolar ratios but did not contain nitrogen. Two sugars were detected which migrated on paper chromatograms with glucose and galactose.     

A hemolytic protein secreted from epidermal cells of the Arabian Gulf catfish, Arius thalassinus (Ruppell)

1. A fright or shock induced toxic secretion (gel) from the epidermis of the Arabian Gulf catfish, Arius thalassinus, exhibits hemolytic activity when tested against red blood cells from many different sources. 2. An enzyme with hemolytic activity, which represents 1.1% of the total soluble gel protein fraction, has been purified to homogeneity. 3. Molecular sieve chromatography and SDS polyacrylamide gel electrophoresis of the purified protein indicate a mol. wt of 34,000. 4. One additional protein component with hemolytic activity was found in the epidermal secretion. 5. Specific activity of the catfish epidermal factor is 20.6 units/mg protein, a level somewhat lower than those of most protein hemolytic factors. 6. The catfish hemolytic factor was not ichthyotoxic when tested against small fish and did not cause lethality when administered intravenously to rabbits.     

Isolation and characterization of a novel polysaccharide as a possible allergen occurring in wheat flour

A new polysaccharide with a molecular weight of 5.0 x 10(4) was isolated as a possible wheat allergen from a water-soluble fraction of flour by affinity chromatography and gel filtration. The isolated polysaccharide was found to be a possible wheat allergen, as it bound specifically to IgE antibodies in the sera of patients allergic to the water-soluble fraction of flour. Chemically, the sugar moiety of the polysaccharide consisted of D-glucose and D-mannose with beta-1,4-linkages in a molar ratio of 4.4:1. Since this mannoglucan is thought to be stable in our body, it would act as a remaining allergen to cause a long-lasting allergic reaction to wheat flour.     

Differentiation of hemagglutinins in tissues infected withChlamydia

Crude, soluble, chlamydial hemagglutinin was prepared from allantoic fluid harvested from embryonated chick eggs and the supernatant fluid of mouse L cells infected with eitherChalamydia psittaci strain 6BC orChlamydia trachomatis strain TW-3. Control nonhemagglutinating specimens of uninfected allantoic fluid and mouse L cells were also prepared. The six preparations were separated by ether-ethanol extraction into lipid-rich and lipid-depleted fractions. Complement-fixing activity was found in the lipid-rich (but not in the lipid-depleted) fraction of infected preparations. In contrast, lipid-rich fractions of infected and uninfected preparations had similar agglutinating activity when sensitive erythrocytes of white Leghorn chickens were used. The lipid-rich fraction of infected and uninfected preparations was separated by thin-layer chromatography (TLC) into seven components with similarR _f values, hemagglutinating patterns, and chemical composition (lipid, protein, and carbohydrate). The highest hemagglutination titers of normal and infected preparations were found in a TLC fraction with similarR _f values and contained lipid, protein, and carbohydrate. This TLC fraction fromC. psittaci 6BC preparations was used in hemagglutination-inhibition studies. The results indicated that chlamydial hemagglutinin extracted by ether-ethanol and separated by TLC contained, in addition to specific hemagglutinin, nonspecific tissue-lipid hemagglutinin(s) identical to that found in normal preparations.     

Chemical and immunological identification of glycolipid-based blood group ABH and Lewis antigens in human kidney

A polar non-acid glycolipid fraction has been isolated from human kidney. It was shown by thin-layer chromatography to be a mixture of glycolipids having more than four carbohydrate residues. Immunological testing revealed strong blood group Lea and A activity together with weak Leb, P1 and B activity. Mass spectrometry of the permethylated and permethylated-reduced (LiAlH4) glycolipid fraction showed that the two major components were a five sugar fucolipid (isomer of Lea) and a glycolipid having four hexoses and one N-acetylhexosamine. In addition, blood group Leb, B and A type hexaglycosylceramides were present. Evidence for small amounts of more complex glycolipids was also found. Acid degradation and gas chromatography of the native fraction revealed fucose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. This is the first chemical isolation and characterization of complex blood group active glycolipids in human kidney. The existence of these molecules is discussed in view of their possible role as transplantation antigens.     

Assay for evaluation of rotavirus-cell interactions: identification of an enterocyte ganglioside fraction that mediates group A porcine rotavirus recognition.

A virus-host cell-binding assay was developed and used to investigate specific binding between group A porcine rotavirus and MA-104 cells or porcine enterocytes. A variety of glycoconjugates and cellular components were screened for their ability to block rotavirus binding to cells. During these experiments a crude ganglioside mixture was observed to specifically block rotavirus binding. On the basis of these results, enterocytes were harvested from susceptible piglets and a polar lipid fraction was isolated by solvent extraction and partitioning. Throughout subsequent purification of this fraction by Sephadex partition, ion-exchange, silicic acid, and thin-layer chromatography, blocking activity behaved as a monosialoganglioside (GMX) that displayed a thin-layer chromatographic mobility between those of GM2 and GM3. The blocking activity of GMX was inhibited by treatment with neuraminidase and ceramide glycanase but not by treatment with protease or heat (100 degrees C). Further purification of GMX by high-pressure liquid chromatography resulted in the resolution of two monosialogangliosides, GMX and a band which comigrated with GM1 on thin-layer chromatography. These data suggest that a cell surface monosialoganglioside or family of monosialogangliosides may function as an in vivo relevant receptor for group A porcine rotavirus and that sialic acid is a required epitope for virus-binding activity.     

Isolation and Properties of Carbohydrate Rich Fraction of Wheat Gluten

Two carbohydrate rich fractions A and B were isolated from wheat gluten. Fraction B contained more lipid than fraction A. Lipid portion of fraction B consisted mainly of glycolipid and was fractionated into five fractions by thin-layer chromatography. The two main fractions were extracted and determined to be galactolipid and glucolipid, respectively, by the analyses of fatty acid and sugar components by gas chromatography. Defatted fraction A was assumed to consist of glycoprotein. After complete pronase digestion of defatted fraction A, the remaining glycopeptide moiety was isolated by column chromatography on DEAE-cellulose followed by gel filtration through Sephadex G–25. The amino acid and sugar components of the glycopeptide were investigated.     

Neuroprotection by Monocarbonyl Dimethoxycurcumin C: Ameliorating the Toxicity of Mutant TDP-43 via HO-1

Mutation of TAR DNA-binding protein-43 (TDP-43) was detected in familiar and sporadic amyotrophic lateral sclerosis, and pathological TDP-43 was identified in the frontotemporal lobar degeneration. The neuroprotective functions of curcumin derivatives were assessed in motor neurons transfected with mutant TDP-43. We found that curcumin derivatives reduced the levels of TDP-43 fragments. Furthermore, we evaluated these compounds on the cellular model that the cells were transfected with TDP-25. We found that the expression level and aggregate formation of TDP-25 were significantly reduced by monocarbonyl dimethoxycurcumin C (Compound C). To study on the neuroprotective functions of curcumin derivatives, the neuroblastoma-spinal cord-34 cells transfected with mutant TDP-43 were assessed by the level of lactate dehydrogenase (LDH) and malondialdehyde bisdimethyl acetal (MDA) that were involved in the oxidative stress. We found that Compound C ameliorated the damage of mutant TDP-43 by reducing the level of MDA and LDH. Furthermore, heme oxygenase-1 (HO-1) was induced by Compound C significantly higher than other compounds. Znpp, which is known an inhibitor of HO-1, dramatically interfered with the function of Compound C. In addition, Compound C was tested in vivo, and HO-1 was significantly upregulated at the hippocampus. These findings suggest that Compound C, which degrades TDP-43 fragment and strengthens the antioxidant ability by HO-1, is a promising agent for TDP-43 proteinopathy.     

Structures of polar lipids from the thermophilic, deep-sea archaeobacterium Methanococcus jannaschii

Cells of Methanococcus jannaschii, grown at 65 degrees C in a defined medium, contained 7% of lipid composed of 87% polar and 13% neutral components. Within the polar fraction 16 lipids were resolved by thin-layer chromatography, 4 of which were present in trace amounts. Staining reactions demonstrated that the more abundant lipids were glycolipids, aminophospholipids, and an aminophosphoglycolipid. Most of the polar fraction (82%) consisted of five diether lipids, which were purified and their structures were resolved largely through nuclear magnetic resonance, mass spectrometry, and optical rotation methods. Macrocyclic diethers had the head groups phosphoethanolamine-(1----6)-beta-D-glucopyranose, beta-D-glucopyranose, and beta-D-glucopyranosyl-(1----6)-beta-D-glucopyranose. Phosphoethanolamine was identified as a head group for both the noncyclized and macrocylic diether core lipids. The neutral lipids were mainly acyclic C30 isoprenoids, predominantly dihydro-, hexahydro, and octahydro-squalenes.