Synthesis and stability of arylsulfatase A and B in fibroblasts from multiple sulfatase deficiency

Fibroblasts from patients with multiple sulfatase deficiency were analyzed for activities of arylsulfatase A and B, iduronate 2-sulfatase and sulfamatase. A group of patients (group I) severely deficient in all sulfatases (residual activities less than or equal to 10% of control) were differentiated from patients (group II) with residual sulfatase activities of up to 90% of control. The synthesis and stability of arylsulfatase A and B were determined in pulse-chase labelling experiments. The apparent rate of synthesis of arylsulfatase A and B varied from 30% to normal in both fibroblasts from group I and II multiple sulfatase deficiency. In group I the molecular activity of the arylsulfatase A and B was more than 10-fold lower than in control fibroblasts. In group II the molecular activity of the arylsulfatase A was twofold to threefold lower and that of arylsulfatase B half of normal. In fibroblasts of both groups the stability of arylsulfatase A polypeptides was significantly diminished. For arylsulfatase B the instability was restricted to the mature 47000-Mr polypeptide and was variable within both groups. These results demonstrate that multiple sulfatase deficiency is a heterogeneous disorder, in which the primary defects can impair both the catalytic properties and the stability of sulfatases.     

Effects of temperature on germination and hyphal growth from ascospores of A-group and B-group Leptosphaeria maculans (phoma stem canker of oilseed rape)

Ascospores of both A‐group and B‐group Leptosphaeria maculans germinated at temperatures from 5–20°C on distilled water agar or detached oilseed rape leaves. After 2 h of incubation on water agar, some A‐group ascospores had germinated at 10–20°C and some B‐group ascospores had germinated at 5–20°C. The percentages of both A‐group and B‐group ascospores that had germinated after 24 h of incubation increased with increasing temperature from 5–20°C. The observed time (Vo₅₀) which elapsed from inoculation until 50% of the spores had germinated was shorter for B‐group than for A‐group ascospores. Germ tube length increased with increasing temperature from 5–20°C for both ascospore groups. Germ tubes from B‐group ascospores were longer than germ tubes from A‐group ascospores at all temperatures tested, but the mean diameter of germ tubes from A‐group ascospores (1.8 μm) was greater than that of those from B‐group ascospores (1.2μm) at 15°C and 20°C. The average number of germ tubes produced from A‐group ascospores (3.8) was greater than that from B‐group ascospores (3.1) after 24 h of incubation at 20°C, on both water agar and leaf surfaces. Germ tubes originated predominantly from interstitial cells or terminal cells of A‐group or B‐group ascospores, respectively, on both water agar and leaf surfaces. Hyphae from A‐group ascospores grew tortuously with extensive branching, whilst those from B‐group ascospores were predominantly long and straight with little branching, whether the ascospores were produced from oilseed rape debris or from crosses between single ascospore isolates, and whether ascospores were germinating on water agar or leaf surfaces.     

Rat antibodies as probes for the characterization of progesterone receptor A and B proteins from laying hen oviduct cytosol

The chicken oviduct contains two different hormone binding forms of the progesterone receptor, A and B. We have prepared rat antisera against both forms of the receptor partially purified from laying hen oviduct. The anti-progesterone receptor A antiserum reacts with both receptor forms on Western blots, while the anti-progesterone receptor B antiserum reacts mainly with the B form. Both antisera also react with the native progesterone receptor proteins as shown by sedimentation analysis of the antibody-receptor complexes. Receptors A and B are recognized on Western blots of total protein from dissolved tissue, indicating that both forms are likely to be physiological components. Epitope mapping experiments show that immunogenicity of both receptor molecules is restricted to structurally related protein domains of 28 kDa in receptor A and of 52 kDa in receptor B.     

Changes in amount and synthesis of poly(A)- and poly(A)+ RNA in growing and resting cultures of Tetrahymena

This investigation deals both qualitatively and quantitatively with the changes of RNA content and synthesis during the culture growth cycle of Tetrahymena. Affinity chromatography with an oligo(dT) column was used to separate poly(A)+ RNA from total RNA. The rates of synthesis of poly(A)- and poly(A)+ RNA were determined in terms of the incorporation of [5-3H]uridine. During the log phase, the cellular RNA and protein contents decreased steadily, whereas during the resting stage, both were constant. The extents of decrease of both fractions of RNA were essentially the same (54.4% and 50.6% for total and poly(A)+ RNA, respectively). Therefore, the relative contents of poly(A)+ RNA was constant from the beginning of the log to the resting stage (4.58%). The decrease in protein content, however, amounted to only 24.8%. Theoretically, a change in the age distribution during culture growth would cause a lower content of both fractions of RNA. The extents of the decrease in the rate of synthesis of both fractions were the same (75% and 79% for poly(A)- and poly(A)+ RNA, respectively). However, this reduction is so large that it cannot be solely the result of a shift of the age distribution of the cell population.     

The Occurrence of Aeromonads in Activated Sludge: Isolation of Aeromonas sobria and its Possible Confusion with Escherichia coli

Strains of Aeromonas have been isolated in high numbers from several activated sludge samples. Immediately after isolation, some displayed only weak or negative oxidase activity, produced both acid and gas from lactose, formed 'metallic'colonies on Endo agar, and had IMViC reactions which were identical with those of Escherichia coli . Although such strains could have been readily confused with E. coli , further biochemical characterization showed that these belonged to the recently-described A. sobria . Nitrogenase activity was absent in all strains of both A. hydrophila and A. sobria . Almost all strains of both taxa were resistant to ampicillin and penicillin V and 67% of both were bete -hemolytic.     

The role of calmodulin in the structure and regulation of phosphorylase kinase from rabbit skeletal muscle.

A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes: (a) from the ribosomal wash and (b) from the postribosomal supernantant. A comparison is made between these preparations with respect to yield and specific activity. eIF-4A and eIF-4D occur mainly in the postribosomal supernatant; eIF-2, eIF-4C and eIF-5 are more evenly divided over both fractions, whereas eIF-1, eIF-3 and eIF-4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF-4A and eIF-4D.     

Relationships of bradyrhizobia from the legumes Apios americana and Desmodium glutinosum.

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with Apios americana, Desmodium glutinosum, and Amphicarpaea bracteata, three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of A. bracteata plants inoculated with isolates from D. glutinosum were equivalent to levels found with native A. bracteata bacterial isolates, but none of the three A. americana isolates tested had high symbiotic effectiveness on A. bracteata. Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both A. americana and D. glutinosum harbored rare bacterial genotypes similar to Bradyrhizobium japonicum USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to Bradyrhizobium elkanii.     

Structures and structure-activity relationships of three mitogenic and complement fixing pectic arabinogalactans from the malian antiulcer plants Cochlospermum tinctorium A. Rich and Vernonia kotschyana Sch. Bip. ex Walp

Structures of three pectic arabinogalactans, one from Vernonia kotschyana (Vk2a) and two from Cochlospermum tinctorium (Ct50A1 and Ct50A2), and their complement fixation and induction of B cell proliferation in vitro were compared. The polysaccharide Vk2a expressed potent biological activity in both assays compared with Ct50A1 and Ct50A2. Vk2a possessed a very high molecular weight (1150 +/- 20 kDa) compared with Ct50A1 and Ct50A2 which both showed a polydisperse nature with the highest molecular weight polymers in each fraction estimated at approximately 105 kDa (Ct1a) and 640 +/- 100 kDa (Ct2a), respectively. The HMW polymers showed complement fixation in the same range as the native fractions. The arabinogalactan II content was low in Vk2a (2%) compared with that in Ct50A1 (23%) and Ct50A2 (12%). The high molecular weight polymers were subjected to digestion with a beta-d-(1, 3)-galactanase-rich fraction from Driselase, oligomers were isolated by HPAEC, and their finer structures were determined by MALDI- and ES-qoToF-MS, linkage, and monosaccharide composition analyses. Vk2a consists of both a galacturonan core and a rhamnogalacturonan core rich in neutral side chains. The backbones of both Ct-polysaccharides consist mainly of RG-I regions with numerous neutral side chains dominated by galactosyl residues, whereas the homogalacturonan regions seem to be small. Differences in the chain lengths of the 6-linked galacto-oligosaccharides attached to the 3-linked galactan core could not be related to the differences in the potencies of the biological activities observed.     

Root hairs and phosphorus acquisition of wheat and barley cultivars

Several genes that restrict nodulation with specific Bradyrhizobiumstrains are known in Glycine max (soybean), and a similar system of nodulation restriction has recently been discovered in the related North American legume Amphicarpaea bracteata. We analyzed how nodulation-restrictive genotypes of each plant interacted with Bradyrhizobium strains sampled from the other host species. Ten bacterial isolates from A. bracteata that nodulated differentially with genotypes of their homologous host legume showed uniform responses to two soybean isogenic lines that differed at the Rj4 locus controlling nodulation restriction: all isolates formed nodules of normal size and morphology on both isolines. However, little or no nitrogen fixation occurred in any of these symbioses. A. bracteata genotypes that displayed broad vs. restricted symbiotic phenotypes toward naturally-associated bradyrhizobia were also tested with two bacterial isolates from soybean (USDA 76 and USDA 123). Both isolates formed nodules and fixed nitrogen in association with both A. bracteata genotypes. However, symbiotic effectiveness (as measured by acetylene reduction assays) was normal only for the combination of USDA 76 with the restrictive A. bracteata genotype. Overall, these results indicate that plant genes that restrict nodulation by certain naturally-associated bradyrhizobia do not confer comparable specificity when plants interact with bacteria from another related legume species.     

Effect of low and high methional concentrations on prostaglandin biosynthesis in microsomes from bovine and sheep vesicular glands.

The effect of methional on prostaglandin biosynthesis from 5,8,11,14-eicosatetraenoic acid was studied with microsomes from both bovine vesicular glands (BVG) and sheep vesicular glands (SVG). Ethylene was identified when methional was added to the fatty acid-microsome incubation systems showing that oxygen centered radicals such as hydroxyl radical were generated during incubation. A low methional level, 1 mM, enhanced the rate of prostaglandin biosynthesis in both BVG and SVG. A high methional level, 10 mM, inhibited prostaglandin biosynthesis in both BVG alone and SVG solubilized with 1% Tween 20. The inhibitory effect of 10 mM methional was reversed by lyophilization. These data suggest that oxygen centered radicals are used in prostaglandin biosynthesis even though they inactivate the enzyme complex.     

Effect of low and high methional concentrations on prostaglandin biosynthesis in microsomes from bovine and sheep vesicular glands

The effect of methional on prostaglandin biosynthesis from 5,8,11, 14-eicosatetraenoic acid was studied with microsomes from both bovine vesicular glands (BVG) and sheep vesicular glands (SVG). Ethylene was identified when methional was added to the fatty acid-microsome incubation systems showing that oxygen centered radicals such as hydroxyl radical were generated during incubation. A low methional level, 1 mM, enhanced the rate of prostaglandin biosynthesis in both BVG and SVG. A high methional level, 10 mM, inhibited prostaglandin biosynthesis in both BVG alone and SVG solubilized with 1% Tween 20. The inhibitory effect of 10 mM methional was reversed by lyophilization. These data suggest that oxygen centered radicals are used in prostaglandin biosynthesis even though they inactivate the enzyme complex.     

Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus

Two genes encoding thermostable xylanases, named xyn10A and xyn11A, from an alkaliphilic Bacillus firmus were cloned and expressed in Escherichia coli. The E. coli harboring either gene showed clear zone with Congo red clearance assay on xylan plate. The Xyn10A and Xyn11A have molecular weights of 45 and 23kDa, respectively, and both show activities on xylan-zymogram. The xyn10A encodes 396 amino acid residues and is very similar to an alkaliphilic xylanase A from alkaliphilic Bacillus halodurans. The Xyn11A contains 210 amino acid residues and only one amino acid different from an endo-beta-1,4-xylanase from B. halodurans. From alignment of the amino acid sequences with other xylanases, Xyn10A and Xyn11A belong to family 10 and 11 glycosyl hydrolases, respectively. Both show activities over the pH range of 4-11 at 37 degrees C and over 80% activities at 70 degrees C. Interestingly both still retain over 70% activities after 16h preincubation at 62 degrees C.     

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.     

Purification, biochemical properties and active sites of N-acetyl-beta-D-hexosaminidases from human seminal plasma.

Two isoenzymes of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) (Hex A and Hex B) from human seminal plasma were purified to homogeneity with specific activities of 26 and 60 units/mg of protein respectively. N-Acetyl-beta-D-glucosaminidase activity was inseparable from N-acetyl-beta-D-galactosaminidase activity in both Hex A and Hex B by various conventional chromatographic procedures. Although Km values of N-acetyl-beta-glucosaminidase activity of Hex A and Hex B were similar (1.33 mM), those of N-acetyl-beta-galactosaminidase activity were 0.14 mM for Hex A and 0.40 mM for Hex B. However, pH optima and temperature optima were identical for N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities of both isoenzymes; Hex A was far more heat-sensitive than Hex B. Thiol-reactive compounds such as silver salts, mercuric salts, p-chloromercuribenzoate and thimerosal strongly inhibited the N-acetyl-beta-glucosaminidase activities of both isoenzymes. GSH protected the enzyme activities from inactivation caused by these reagents, confirming the presence of thiol groups at the active centres. Inhibitions of N-acetyl-beta-glucosaminidase activities of both isoenzymes by metal salts and organic anions were comparable; acetate and arsenite were effective inhibitors for both isoenzymes. In contrast, inhibitions of N-acetyl-beta-glucosaminidase activities of the two isoenzymes by iodoacetic acid, iodoacetamide and ethylmaleimide were not comparable; Hex B was more susceptible to inhibition by these agents at 20 mM concentration. The N-acetyl-beta-glucosaminidase activities of both isoenzymes are strongly inhibited, in decreasing order, by N-acetyl-galactosamine, mannosamine, disaccharic acid lactone, N-acetylglucosamine and gluconolactone. The Ki values of the N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities for N-acetylhexosamines and results from mixed-substrate kinetics indicated that the activities for the two substrates are located at different sites in Hex A and at the same site in Hex B. The Mr values of Hex A and Hex B were determined to be 195,000 and 210,000 respectively by gel filtration through Sephadex G-200. SDS/polyacrylamide-gel electrophoresis revealed that Hex A and Hex B are each composed of four subunits corresponding to Mr about 50,000 each. No further polypeptide chain was obtained after reduction and alkylation of Hex A and Hex B with 10 mM-dithiothreitol and 10 mM-iodoacetamide.     

Short-term and long-term root respiratory acclimation to elevated temperatures associated with root thermotolerance for two Agrostis grass species

This study was designed to investigate whether thermotolerant roots exhibit respiratory acclimation to elevated temperatures. Root respiratory acclimation traits in response to increasing temperatures were compared between two Agrostis species contrasting in heat tolerance: thermal A. scabra and heat-sensitive A. stolonifera. Roots of both species were exposed to 17, 27, or 37 degrees C. Root RGR declined with increasing temperatures from 17 degrees C to 37 degrees C in both species; however, root growth of A. scabra maintained a significantly higher RGR than A. stolonifera at 27 degrees C or 37 degrees C. A. scabra exhibited a significantly higher respiration acclimation potential to elevated temperatures, both in the short term (60 min) and in the long term (7-28 d) as compared with A. stolonifera, when temperatures increased from 17 degrees C to 27 degrees C or from 27 degrees C to 37 degrees C. Thermal A. scabra also maintained a significantly lower maintenance cost than A. stolonifera as temperatures increased to 27 degrees C or 37 degrees C. The results suggested that root thermotolerance of thermal A. scabra was associated with both short-term and long-term respiratory acclimation to changes in temperatures. The superior ability of adjusting the rate of root respiration to compensate for increases in carbon demand during short- or long-term temperature increases in the heat-tolerant A. scabra may result in the reduction in carbon expenditure or costs for maintenance, leading to extended root survivability in high temperature soils.     

Omeprazole, an inducer of human CYP1A1 and 1A2, is not a ligand for the Ah receptor.

Omeprazole is a benzimidazole derivative which induces both P450 1A1 and 1A2 in human liver in vitro and in vivo. Northern blot analysis of polyA RNA prepared from primary cultures of human hepatocytes indicates that both 1A1 and 1A2 messages are induced by beta-naphthoflavone and omeprazole. Co-treatment of cells with these inducers and with actinomycin D or cycloheximide results in no accumulation of both mRNA or superinduction of 1A1 mRNA, respectively. 9S enriched fraction of cytosol was prepared either from human hepatocytes in culture or from human liver tissue and analyzed by sucrose density gradient sedimentation for its capacity to bind 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), omeprazole or omeprazole sulfone (a metabolite of omeprazole in man). Whereas 2 microM TCDD displaced almost totally [3H]TCDD from the Ah receptor, both omeprazole and omeprazole sulfone did not, even at 5000-fold molar excess. In addition, when [14C] omeprazole was incubated with 9S enriched fraction of human liver or hepatocyte cytosol, no interaction could be detected in sucrose density gradient. These experiments suggest that omeprazole is not a ligand for the human liver Ah receptor.     

Comparative molecular phylogeography of North American softshell turtles (Apalone): implications for regional and wide-scale historical evolutionary forces

We use a comparative analysis of partial cytochrome b sequences to evaluate the evolutionary forces shaping wide-scale phylogeographic patterns of all three North American softshell turtles (Apalone ferox, A. mutica, and A. spinifera). The overall phylogeographic patterns are concordant with results from both extensive regional studies of southeastern species, implicating historical vicariant processes during the Pliocene and Pleistocene, and investigations of more northerly distributed species, indicating a bottleneck effect of recent dispersal into postglacial habitat. We also resolved a novel, shared genetic break between northern-western and southeastern populations within both A. mutica and A. spinifera, demonstrating the value of using widespread taxa to evaluate both regional and wider scale phylogeographic patterns. The extensive phylogenetic structure and sequence divergences within both A. mutica and A. spinifera contrast sharply with most previous studies of turtles and with the hypothesis that turtles in general have slow rates of mtDNA evolution.     

草鱼出血病混合感染的嗜水气单胞菌的分离、鉴定与理化特性

从患出血病草鱼的肝脏病灶中分离筛选出2株致病菌。取病鱼样品组织过滤液接种CIK细胞、培养, 电镜下观察到细胞质中含有草鱼呼肠孤病毒样颗粒和包涵体, 病毒颗粒大小65 nm~ 70 nm, 包涵体0.46 μm~1.81 μm。人工回归感染实验显示分离的菌株及细胞毒悬液均能使草鱼致病死亡。对分离菌株进行细胞形态学、理化特性分析及药敏试验, 初步判定所分离的2株菌均为嗜水气单胞菌。进一步对菌株进行DNA分子鉴定, 结果显示2株菌的16S rRNA基因、促旋酶亚单位蛋白(gryB)基因均与GenBank上的嗜水     

Habitat and species differences in prevalence and intensity of Neobenedenia melleni (Monogenea: Capsalidae) on sympatric Caribbean surgeonfishes (Acanthuridae)

We quantified Neobenedenia melleni from the skin of Caribbean surgeonfishes (Acanthuridae) from June through October 2005 and 2007. Prevalence, or mean intensity of infection, or both, varied significantly among the 3 species, and among sites and between years for the most heavily infected species, blue tang (Acanthurus coeruleus). Among 6 sites sampled, no more than 12% of ocean surgeonfish (Acanthurus bahianus) were infected, compared with 10 to 100% of A. coeruleus. The prevalence of infection among doctorfish (Acanthurus chirurgus), collected at only 1 of the sites, was intermediate between the other 2 species (46%). Mean intensity (range) of infection for the few infected A. bahianus was 1 (1) to 3 (1-8), compared with 1.3 (1-2) to 14.3 (1-59) for A. coeruleus, and 2.5 (1-8) for A. chirurgus. Expected abundance of N. melleni on A. coeruleus from shallow bay sites was greater than for those from non-bay sites. Higher infections on A. coeruleus may be attributable to differences in habitat use, or susceptibility to infection, or both, compared to other species. Among site and between-year differences may be associated with differences in benthic habitat, or water conditions, or both. This system seems ideal for future comparative studies on the relationship between environmental variables and parasites on Caribbean coral reefs.     

Multiple sulfatase deficiency: degradation of arylsulfatase A and B after endocytosis in fibroblasts

Multiple sulfatase deficiency can be classified into group I with severe and group II with moderate deficiencies in sulfatases. In fibroblasts in both groups the stability of arylsulfatase A and of the 47000-Mr form of arylsulfatase B is decreased [F. Steckel, A. Hasilik & K. von Figura (1985) Eur. J. Biochem. 151, 141-145]. After endocytosis in control fibroblasts or those from multiple sulfatase deficiency, arylsulfatase A and B derived from the latter were subjected to enhanced degradation in both types of recipient cells. The degradation was closely linked in time to endocytosis. Whereas instability of arylsulfatase A derived from different cell lines from multiple sulfatase deficiency was comparable, a marked heterogeneity was observed for the instability of the 47000-Mr polypeptide of arylsulfatase B. Each of the cell lines from multiple sulfatase deficiency synthesized arylsulfatase A and B polypeptides with normal and with decreased stability. Treatment with benzyloxycarbonyl-Phe-Ala-CHN2, an inhibitor of cysteine proteinases, stabilized arylsulfatase A polypeptides and partially restored arylsulfatase A activity in group II fibroblasts. The inhibitor had no protective effect on the 47000-Mr polypeptide or the activity of arylsulfatase B. The bearing of these findings on the yet unknown primary defect in multiple sulfatase deficiency is discussed.