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1.
Using an immunoblotting technique and goat antihuman C4, we observed five distinct electrophoretic variants of C4 in a panel of 60 random dogs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated C4 showed that dog C4 is composed of three polypeptide subunit chains (, , and ) and that structural variability occurs within the - and -chain regions. Two distinct molecular weight forms of both the C4- ( A and B) and C4-( A and B) chain were detected. The variant forms of C4 and C4 were found in association with particular C4 allotypes.  相似文献   

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In an attempt to relate structure to anticoccidial activity, a number of 5-modified analogs of 4-deoxypyridoxol (4-DOP) and α4-norpyridoxol have been synthesized and their biological activities examined. The compounds prepared include the 5-(3-hydroxypropyl), 5-(2-hydroxyethyl), 5-(1-hydroxyethyl), formyl and acetyl analogs of 4-DOP, and 5-(3-hydroxypropyl), formyl, ethoxycarbonyl, carbamoyl and hydroxyl analogs of α4-norpyridoxol. Among these compounds, 4-deoxyisopyridoxal and α4-norisopyridoxal were found to exhibit anticoccidal activity.  相似文献   

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The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na(+) flux assay revealed that α4β2 formed Na(+) permeable channels in lipid vesicles. Efflux of Na(+) through the α4β2 channels reduced intra-vesicle Sodium Green? fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators.  相似文献   

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Enterotoxigenic Escherichia coli (ETEC) with fimbriae of the F4 family are one of the major causes of diarrhea and death among neonatal and young piglets. Bacteria use the F4 fimbriae to adhere to specific receptors expressed on the surface of the enterocytes. F4 fimbriae exist in three different antigenic variants, F4ab, F4ac, and F4ad, of which F4ac is the most common. Resistance to ETEC F4ab/F4ac adhesion in pigs has been shown to be inherited as an autosomal recessive trait. In previous studies the ETEC F4ab/F4ac receptor locus (F4bcR) was mapped to the q41 region on pig chromosome 13. A polymorphism within an intron of the mucin 4 (MUC4) gene, which is one of the possible candidate genes located in this region, was shown earlier to cosegregate with the F4bcR alleles. Recently, we discovered a Large White boar from a Swiss experimental herd with a recombination between F4bcR and MUC4. A three?Cgeneration pedigree including 45 offspring was generated with the aim to use this recombination event to refine the localization of the F4bcR locus. All pigs were phenotyped using the microscopic adhesion test and genotyped for a total of 59 markers. The recombination event was mapped to a 220-kb region between a newly detected SNP in the leishmanolysin-like gene (LMLN g.15920) and SNP ALGA0072075. In this study the six SNPs ALGA0072075, ALGA0106330, MUC13-226, MUC13-813, DIA0000584, and MARC0006918 were in complete linkage disequilibrium with F4bcR. Based on this finding and earlier investigations, we suggest that the locus for F4bcR is located between the LMLN locus and microsatellite S0283.  相似文献   

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A previous study on the genetics of mouse C4-binding protein (C4-bp) indicated the existence of a genetic polymorphism. Two genetic variants were reported and their segregation used to determine the mapping position of the C4BP locus to the H-2D-Qa interval of the mouse H-2 system. We show here, however, that purified C4-bp does not display the previously reported polymorphism. The mapping position of C4BP in the mouse therefore remains undetermined.  相似文献   

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The article comprises three subsequently published studies of the taxonomy of Cuban cyanophyte/cyanobacterial flora: (4)Lyngbyopsis willei: This oscillatorialean genus and species, described byGardner (1927) from mountain creeks in Puerto Rico, was found more than 50 years later in similar localities in Cuba. The morphological variability of the Cuban populations is described and similarity with the genusSchizothrix (sect.Inactis) discussed. —(5)Cylindrospermum-species: The morphological variability of twenty-oneCylindrospermum-populations collected in Cuba was studied, documented by graphical methods and compared with the published data. Four new taxa were recognized (C. minutissimum v.rinoi, C. zonatum, C. bourrellyi, andC. desikacharyi). The other populations belong to the variation ranges ofC. breve, C. minutissimum, C. michailovskoense, andC. muscicola v.kashmiriense.— (6)Gomphosphaerioideae-species: Fifty eight populations (9 species) of the subfamilyGomphosphaerioideae (Microcystaceae, Chroococcales) from freshwater biotopes of Cuba were evaluated: Four planktic species with a probable cosmopolitan distribution were found (Coelosphaerium kuetzinginianum, C. minutissimum, Snowella lacustris andCoelomoron pusillus), and from the genusCoelomoron Buell two new species,C. microcystoides andC. vestitus were described. The tropical planktic speciesWoronichinia fremyi forms occasionally water blooms in larger reservoirs. Two tropical species from the genusGomphosphaeria Kütz, were recognized,G. multiplex (Nyg.) c. n. andG. semen-vitis sp. n.  相似文献   

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In this paper we construct a mathematical model for DNA replication based on Shannon's mathematical theory for communication. We treat DNA replication as a communication channel. We show that the mean replication rate is maximal with four nucleotide bases under the primary assumption that the pairing time of the G–C bases is between 1.65 and 3 times the pairing time of the A–T bases.  相似文献   

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A bacterial strain (strain S5) which grows aerobically with the sulfonated azo compound 4-carboxy-4′-sulfoazobenzene as the sole source of carbon and energy was isolated. This strain was obtained by continuous adaptation of “Hydrogenophaga palleronii” S1, which has the ability to grow aerobically with 4-aminobenzenesulfonate. Strain S5 probably cleaves 4-carboxy-4′-sulfoazobenzene reductively under aerobic conditions to 4-aminobenzoate and 4-aminobenzene-sulfonate, which are mineralized by previously established degradation pathways.It is generally assumed that sulfonated azo dyes are not degraded under aerobic conditions (14). Nevertheless, there have been some reports which suggest a conversion of certain sulfonated azo dyes under aerobic conditions (3, 7, 8, 13, 15). Furthermore, certain carboxylated analogs of sulfonated azo compounds are utilized aerobically as the sole source of carbon and energy by specifically adapted bacteria (11, 12, 16, 17). However, unequivocal evidence for the productive mineralization of a sulfonated azo compound by bacteria is lacking. In the present article the first observation of the utilization of a sulfonated azo compound as the sole source of carbon and energy by a bacterial strain is reported.Previously, a mixed bacterial culture which mineralizes sulfanilate (4-aminobenzenesulfonate) was isolated. This coculture consisted of the strains “Hydrogenophaga palleronii” S1 and Agrobacterium radiobacter S2 (4, 5). Because sulfanilate occurs as an azoaryl structural element in many azo dyes, it was of interest whether this mixed culture could adopt the ability to reduce azo bonds and release sulfanilate as growth substrate. Therefore, the model sulfonated azo compound 4-carboxy-4′-sulfoazobenzene (CSAB) was synthesized by nitro-amine condensation starting with sulfanilic acid and 4-nitrobenzoic acid (1). The precipitated CSAB was separated from the reaction mixture by filtration and purified by repeated dissolution in alkali and precipitation with acid. The identity and purity of the bright orange product were analyzed by UV-visible light spectroscopy, elementary analysis, and high-pressure liquid chromatography (HPLC). For the solid material obtained, molar extinction coefficients of 23.74 and 1.13 mM−1 cm−1 in water were determined at the wavelengths of 326 and 434 nm, respectively. The elementary analytic results were consistent with the structure of CSAB. The purity of the preparation was tested by HPLC with a reversed-phase column and a solvent gradient from 1 to 90% (vol/vol) methanol and 0.3% (vol/vol) H3PO4. A single band which showed absorbance at a wavelength of 326 nm was eluted. At 210 nm a minor contaminant (about 15% of the signal intensity of CSAB) was detected. This compound was clearly different from either 4-nitrobenzoate or sulfanilate.The mixed culture was grown in repeated batch cultures in a mineral medium with sulfanilate (5 mM). About every 2 weeks the culture was transferred (1:10 [vol/vol]) to fresh medium, in which the sulfanilate concentration was subsequently reduced and the CSAB concentration increased (±0.5 mM each). The color of the azo dye disappeared after 2 months. The culture was transferred to a solid mineral medium with CSAB as the sole source of carbon. From this culture was obtained strain S5, which grew aerobically with the sulfonated azo compound CSAB as its sole source of carbon and energy and with a doubling time of 9.5 h (Fig. (Fig.1).1). The complete disappearance of the dye was demonstrated by the loss of the orange color from the medium and by HPLC analysis, whereas CSAB was not degraded in a sterile control flask. Based on its colony morphology and the results obtained with the commercial identification system Biolog GN, this strain strongly resembled “H. palleronii” S1. Recently, it was demonstrated that, in the presence of low concentrations of biotin, cyanocobalamin, and 4-aminobenzoate, strain S1 also grows in axenic culture with sulfanilate (2). Therefore the adaptation experiment was repeated in the presence of these three substances with a pure culture of strain S1. This experiment also resulted in the isolation of a strain which grew in axenic culture with CSAB as the sole source of carbon and energy. Open in a separate windowFIG. 1Aerobic growth of strain S5 with CSAB as the sole source of carbon and energy. The growth was determined photometrically (OD546), and the turnover of CSAB was measured by HPLC with a reversed-phase column and a solvent gradient consisting of H2O, methanol, and 0.3% H3PO4 with increasing concentrations of methanol (1 to 90%). An OD546 of 1 corresponded to 0.33 mg of protein ml−1.To ensure that the genetic backgrounds of strains S5 and S1 were identical, the genes for the 16S rRNAs were amplified by PCR with different universal primers (6) and sequenced in comparison to the corresponding gene from the type strain, H. palleronii DSM 63. It was found that the sequences from strains S1 and S5 were > 99.8% identical (there were only two discrepancies between the two sequences), but they showed only 97.7 to 97.9% identity with the 16S rRNA gene from H. palleronii DSM 63. It was therefore concluded that strain S5 was derived from strain S1 and that the strains do not belong to the species H. palleronii.A reductive cleavage of the azo bond of CSAB would result in the formation of 4-aminobenzoate and sulfanilate. Like the parent strain, S1, strain S5 grew in the presence of sulfanilate, 4-aminobenzoate, and 4-sulfocatechol. The doubling times with these compounds were 6.2 to 6.4 h. We therefore investigated whether reductive cleavage of CSAB by strain S5 occurs. Strain S5 was grown aerobically with 5 mM CSAB, and cell extracts were prepared (10) in different buffers. These cell extracts were incubated aerobically in cuvettes containing 50 mM Tris-HCl buffer (pH 8.0), 0.5 mM CSAB, 1 mM NADH, or 1 mM NADPH and with various mixtures of possible cofactors. The enzyme activity was measured spectrophotometrically at the absorption maximum for CSAB (at a wavelength of 434 nm), but no significant decrease in absorbance was observed. Neither addition of a membrane fraction nor performing the enzyme assays under anaerobic conditions (9) improved the turnover of CSAB in the cell-free system. Furthermore, there was no significant increase in azo reductase activity when harvested cells were resuspended in the culture supernatant instead of Tris-HCl buffer.The maximal enzyme activities observed for cell extracts were only about 30% of the activities found for intact cells. This suggested that during the disruption of the cells some important components of the azo reductase system were destroyed or some cofactors were present in only limiting quantities.Because it was difficult to obtain reproducible enzyme activities with cell extracts, the turnover of CSAB by resting cells was investigated. Cells of strain S5 were grown with CSAB (5 mM), harvested by centrifugation, resuspended in Tris-HCl at an optical density at 546 nm (OD546) of 5.3, and incubated in a water bath shaker (140 rpm; 30°C) with 0.5 mM CSAB (Fig. (Fig.2).2). Thus, the transient accumulation of two metabolites in the supernatants was observed by reversed-phase HPLC (column size, 250 by 4.6 mm) (SIL 100; Grom, Herrenberg, Germany). The solvent system consisted of a solvent gradient with increasing concentrations of methanol, starting with 1% (vol/vol) methanol, 98.9% (vol/vol) water, and 0.1% H3PO4. The flow rate was 0.7 ml min−1. The metabolites formed were identified as sulfanilate and 4-sulfocatechol by comparison of their retention times and in situ UV-visible light-spectra with authentic standards. Surprisingly, the concentration of 4-sulfocatechol in the medium increased (and decreased) during the experiment more rapidly than the concentration of sulfanilate (Fig. (Fig.2).2). 4-Sulfocatechol also temporarily accumulated when resting cells of strain S1 were incubated with sulfanilate (4, 5). This suggested that in the resting-cell assay the initial activity of the sulfanilate-converting enzyme was higher than the activity of the 4-sulfocatechol-oxidizing enzyme protocatechuate-3,4-dioxygenase type II. Presumably, the activity of the sulfanilate-converting enzyme decreased during the experiment more rapidly than the activity of protocatechuate-3,4-dioxygenase type II. No accumulation of 4-aminobenzoate or protocatechuate was found by HPLC analysis during the experiment. In a control experiment with cells of strain S1 grown with 4-aminobenzenesulfonate, no turnover of CSAB was observed by HPLC analysis. Open in a separate windowFIG. 2Conversion of CSAB (•) to sulfanilate (▪) and 4-sulfocatechol (□) by resting cells of strain S5. Strain S5 was grown in a mineral medium with CSAB as the sole source of carbon and energy, and resting cells were prepared as described in the text.The detection of sulfanilate derived from CSAB suggested a reductive cleavage of CSAB, yielding sulfanilate as one of the reduction products. This reaction should also proceed in the absence of oxygen. Therefore, resting cells were incubated under anaerobic conditions with CSAB. Surprisingly, the rate of CSAB turnover under anaerobic conditions was <2% of the turnover rate under aerobic conditions.A further indication of a reductive cleavage of CSAB into sulfanilate and 4-aminobenzoate was obtained by growing strain S5 with CSAB or a complex medium (HPG medium) (4). When the cells were grown in a mineral medium with CSAB and the turnover of the substrates was analyzed by HPLC, it was found that resting cells converted CSAB, 4-aminobenzoate, or 4-aminobenzenesulfonate with specific activities of 0.012, 0.026, and 0.011 μmol min−1 mg of protein−1, respectively. In contrast, after growth of the cells in HPG medium, these activities were only 0.007, 0.010, and 0.003 μmol min−1 mg of protein−1, respectively. Incubation of resting cells with CSAB and different potential inhibitors of ring cleavage dioxygenases showed that the turnover of CSAB was almost completely inhibited by the addition of 8-hydroxyquinoline or 2,2′-bipyridyl (1 mM each). The presence of 4-nitrocatechol (0.25 mM) also resulted in a pronounced reduction of the rate of CSAB turnover (6% of the rate in the absence of the inhibitor). In this system as well the formation of 4-sulfocatechol was observed.The degradation of sulfanilate and 4-aminobenzoate by strain S1 has been previously studied (5). The proposed degradation pathway for CSAB and its reduction products is shown in Fig. Fig.3.3. Open in a separate windowFIG. 3Proposed pathway for the degradation of CSAB by strain S5. 4AB, 4-aminobenzoate; 4ABS, 4-aminobenzenesulfonate (sulfanilate); 3,4DHB, 3,4-dihydroxybenzoate (protocatechuate); 4SC, 4-sulfocatechol; 2H4CMSA, 2-hydroxy-4-carboxymuconic semialdehyde; 3SM, 3-sulfomuconate; 4SL, 4-carboxymethyl-4-sulfobut-2-en-4-olide (4-sulfolactone); MA, maleylacetate; 3OA, 3-oxoadipate; TCC, tricarboxylic acid cycle.To obtain some information about the substrate specificity, resting cells were incubated with CSAB, 4,4′-dicarboxyazobenzene (DCAB), 4-hydroxy-4′-sulfoazobenzene, methyl orange [4-(N,N-dimethyl)-4′-sulfoazobenzene; color index (C.I.) 13025], orange II {4-[(2-hydroxy-1-naphthalenyl)azo]-benzenesulfonic acid; C.I. 15510}, or sunset yellow FCF {6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthol-6-sulfonic acid; FD&C no. 6; C.I. 15985}. Of these compounds, only CSAB and DCAB were converted by resting cells. DCAB was also utilized by strain S5 as the sole source of carbon and energy. Furthermore, no growth of strain S5 was found with acid black 24 and 52, acid blue 113, acid red 1, amaranth, direct red 81, direct yellow 4 and 50, mordant yellow 3, and naphthol blue black.The results presented in this study suggest that bacterial cultures with the ability to aerobically degrade simple sulfonated azo dyes may be obtained after preadaptation to sulfonated aminoaromatics and/or when reductive cleavage of the azo bond gives rise to an aerobically assimilable aminoaromatic structure, like 4-aminobenzoate. This selection scheme circumvents the problems observed during attempts to adapt bacteria with the ability to degrade carboxylated azo compounds for the degradation of sulfonated azo compounds (12). The ability of strain S5 to mineralize CSAB suggests that it is possible to degrade sulfonated azo dyes under aerobic conditions if biological systems which can grow and can mineralize the reduction products are available.

Nucleotide sequence accession number.

The nucleotide sequences for the 16S rRNAs from strains S5 and S1 have been deposited in the GenBank data library under accession no. AF019037 and AF019073, respectively.  相似文献   

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4-Nitrophenyl penta-N-acetyl-β-chitopentaoside [(GlcNAc)5-pNP] was hydrolyzed by a family GH-19 class II barley chitinase, and the enzymatic reaction was monitored by real-time ESIMS. The wild-type enzyme hydrolyzed (GlcNAc)5-pNP producing predominantly (GlcNAc)3-pNP and a lesser amount of (GlcNAc)2-pNP, indicating that the (GlcNAc)5 portion of the substrate binds predominantly to subsites −2 ∼ +3 and less frequently to −3 ∼ +2. However, (GlcNAc)2-pNP was mainly produced from (GlcNAc)5-pNP by mutated enzymes, in which Trp72 and Trp82 located at +3/+4 were substituted with alanine (W72A and W72A/W82A), indicating that the (GlcNAc)5 portion of the substrate binds predominantly to subsites −3 ∼ +2 of the mutants. The mutations of the tryptophan residues resulted in a significant shift of the substrate-binding mode to the glycon side, supporting the idea that the indole side chain of Trp72 interacts with the 4-nitrophenyl moiety of the substrate at subsite +4.  相似文献   

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The α4 subunit of the GABAA receptor (GABAAR) is highly expressed in the thalamus where receptors containing the α4 and δ subunits are major mediators of tonic inhibition. The α4 subunit also exhibits considerable plasticity in a number of physiological and pathological conditions, raising questions about the expression of remaining GABAAR subunits when the α4 subunit is absent. Immunohistochemical studies of an α4 subunit knockout (KO) mouse revealed a substantial decrease in δ subunit expression in the ventrobasal nucleus of the thalamus as well as other forebrain regions where the α4 subunit is normally expressed. In contrast, several subunits associated primarily with phasic inhibition, including the α1 and γ2 subunits, were moderately increased. Intracellular localization of the δ subunit was also altered. While δ subunit labeling was decreased within the neuropil, some labeling remained in the cell bodies of many neurons in the ventrobasal nucleus. Confocal microscopy demonstrated co-localization of this labeling with an endoplasmic reticulum marker, and electron microscopy demonstrated increased immunogold labeling near the endoplasmic reticulum in the α4 KO mouse. These results emphasize the strong partnership of the δ and α4 subunit in the thalamus and suggest that the α4 subunit of the GABAAR plays a critical role in trafficking of the δ subunit to the neuronal surface. The findings also suggest that previously observed reductions in tonic inhibition in the α4 subunit KO mouse are likely to be related to alterations in δ subunit expression, in addition to loss of the α4 subunit.  相似文献   

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The long loop connecting transmembrane α4 and α5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro193Pro194_Pro196) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH 9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the α4–α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4–α5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro193. Further MD simulations revealed that the most critical residue−Pro193 for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa α4–α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity.  相似文献   

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