Isogenic P-fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of α Gal(1–4)β Gal binding in virulence of a wild-type strain

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap⁺, sfa⁺, pil⁺, hly⁺ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were sub-cloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (Nal^R) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA⁺ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or α Gal(1–4)β Gal-coated latex beads. CBA mice (N =100) were challenged transurethrally with 10⁵, 10⁶, 10⁷, or 10⁹ cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uro-pathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.     

Structural Analysis of the Sixth Immunoglobulin-Like Domain of Mouse Neural Cell Adhesion Molecule L1 and Its Interactions with αvβ3, αIIbβ3, and α5β1 Integrins

Abstract: Previous experiments suggested that the human cell adhesion molecule L1 interacts with different integrins via its sixth immunoglobulin-like domain in an RGD-dependent manner. Here we have described the expression of this domain from early postnatal mouse brain, analyzed the structure of the recombinant protein by circular dichroism and fluorescence spectroscopy, and performed solid-phase binding studies to αvβ3, αIIbβ3, and α5β1 integrins. The domain was found to have the expected β-sheet organization, which was lost in the presence of guanidine hydrochloride. The midpoint of the single-step transition occurred at 1.5 M guanidine hydrochloride. The sixth immunoglobulin-like domain of mouse brain L1 contains two RGD motifs and was found to bind in a concentration-dependent and saturable way to αvβ3, αIIbβ3, and α5β1 integrins, suggesting specific interactions with these ligands. However, only the interaction to αvβ3 could be inhibited in a concentration-dependent manner by an RGD-containing peptide, and the IC₅₀ was determined to be ∼20 n M . Mutants of the domain, which lack either one or both of the RGD sites, demonstrated that the RGD site comprising residues 562–564 is involved in the interaction to αvβ3. Our findings indicate an RGD-independent mechanism for the interactions to αIIbβ3 and α5β1, as no involvement of any RGD motif could be demonstrated.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Hypolipidaemic Effects of (24R)-4α-methyl-5α-stigmasta-7,22-dien-3β-ol Derived from Aurantiochytrium mangrovei BT3 in the HEPG2 Cell Line

Applied Biochemistry and Microbiology - Aurantiochytrium mangrovei BT3, a heterotrophic marine microalga, has the ability to produce high amounts of polyunsaturated fatty acids and bioactive...     

异种移植中Galα(1,3)Gal抗原的研究近况

同种异体组织和器官移植物供体来源有限,使得异种移植再度成为移植领域的研究热点。异种移植的主要障碍是人体内存在的天然抗体与移植物表面含有α1,3半乳糖残基[Galα(1,3)Gal,αGal]的抗原结合,激活补体系统和炎症反应,导致超急性移植排斥反应(HAR)的发生,使移植物失活。除人类和旧世纪猴外,其它所有哺乳动物的体内都含有αGal抗原,该抗原是由一组具有Galα(1,3)Gal双糖末端的糖蛋白或糖脂组成的,它的形成依赖于α1,3半乳糖基转移酶(αGT)的催化。目前,针对αGal抗原克服超急性移植排斥反应的方法主要有如下几种：(1)酶处理去除内皮细胞表面的αGal抗原;(2)物理化学方法去除人体血浆中存在的特异性天然抗体;(3)基因工程方法改造表达催化αGal抗原形成的相关酶基因,从而影响该抗原的表达。     

Tandem repeats of the tetramer 5'-CAAT-3'present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae

A novel lipopolysaccharide (LPS) biosynthesis gene, lic2B, which is required for the biosynthesis of a phase-variable LPS structure expressed by Haemo philus influenzae RM7004 is described. The product of this gene is homologous to Lic2A and the recently described LPS biosynthetic enzymes, LgtB from Neis seria gonorrhoea and LgtE from Neisseria meningitidis, and LpsA from Pasteurella haemolytica. Of this family of enzymes only Lic2A contains the repetitive tetrapeptide motif (SINQ)ⁿ encoded by multiple tandem repeats of 5′-CAAT-3′. This observation suggested that (SINQ)ⁿ might not be a prerequisite for the catalytic activity of this protein. To address this hypothesis, we deleted the 5′-CAAT-3’repeats from lic2A so that the protein encoded by the modified gene was analogous to Lic2B. This mutation had no apparent effect on the overall apparent molecular weight of LPS as judged by Tricine-SDS-PAGE and did not affect ability to react with monoclonal antibody 4C4. It was therefore concluded that (SINQ)ⁿ is not a prerequisite for the enzymatic function of Lic2A and that the 5′-CAAT-3’repeats in lic2A function solely as a mechan ism for generating phase variation. This observation suggested that wide variation in the number of 5’-CAAT-3’repeats might be tolerated in lic2A, and this was confirmed by surveying the number of 5′-CAAT-3’repeats in a range of different H. influenzae strains. The predicted secondary structure of (SINQ)ⁿ indicates that it forms a highly flexible random coiled structure, which is unlikely to impede formation of the domains that may be required for catalytic activity. This characteristic is also a feature of repetitive tetrapeptides encoded by other tetrameric repeats located within coding sequences present on the chromosome of H. influenzae Rd.     

The loop between β-strands β2 and β3 and its interaction with the N-terminal α-helix is essential for biogenesis of α7 nicotinic receptors

Recently, we have shown that the α-helix present at the N-termini of α7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking β-strands β2 and β3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant α7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (α3β4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric α7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the α-helix is an important requirement for receptor assembly.     

Use of chromosomal gene fusions to investigate the role of repetitive DNA in regulation of genes involved in lipopolysaccharide biosynthesis in Haemophilus influenzae.

The lic3 locus of Haemophilus influenzae consists of four open reading frames. The derived amino acid sequences of orf2 and orf4 exhibit homology to Escherichia coli GalE and AdK, respectively. The functions of orf1 and orf3 remain unknown. orf1 contains multiple tandem repeats of the tetrameric DNA sequence CAAT near the 5' end. Two possible translational starts (ATG1 and ATG2) lie upstream. We have used lacZ fusions to investigate whether changes in the number of CAAT repeats in conjunction with differential usage of the upstream frames control the expression of lic3-orf1. Phase-variable expression of lacZ was observed for individual colonies and could be related to variable numbers of CAAT repeats. Of the three possible upstream frames, only one, containing the more downstream of the two possible ATG start codons (ATG2), is used for strong expression of lacZ. Utilization of the more upstream ATG (ATG1) or ATG2 was observed with medium-level expression, while utilization of any of the three possible frames was observed when lacZ was expressed at low to undetectable levels, indicating that other mechanisms may affect expression. To investigate this, lacZ was fused in frame with ATG2 of lic3-orf1, with concomitant deletion of the repeats. Phase-variable expression was still observed, supporting the view that an alternative level of control operates in conjunction with the repeat mechanism.     

The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence βGalNAc(1–4)βGal found in glycosphingolipids asialo-GM1 and asialo-GM2

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. The pilus adhesin of P. aeruginosa strains PAK and PAO has been shown to bind to the glycolipid asialo-GM₁ (Lee et al., 1994 —accompanying article). PAK and PAO pili were examined for their abilities to bind to the synthetic βGalNAc(1–4)βGal (a minimal structural carbohydrate receptor sequence of asialo-GM₁ and asialo-GM₂ proposed by Krivan et al., 1988a) using solid-phase binding assays. Both pill specifically bound to βGalNAc(1–4)βGal. The binding of βGal-NAc(1–4)βGal-Biotin to the Immobilized PAK and PAO pili was inhibited by corresponding free pili. The receptor binding domain of the PAK pilus resides in the C-terminal disulphide-looped region (residues 128–144) of the pilin structural subunit (Irvin et al., 1989). Biotinylated synthetic peptides corresponding the C-terminal residues 128–144 of P. aeruginosa PAK and PAO pilin molecules were shown to bind to the βGalNAc(1–4)βGal-(bovine serum albumin (BSA)). The binding of biotinylated peptides to βGalNAc-(1–4)βGal-BSA was inhibited by PAK pili, Ac-KCTSDQDEOFIPKGCSK-OH (AcPAK(128–144)_ox-OH) and Ac-ACKSTQDPMFTPKGCDN-OH (AcPAO(128–144)_ox-OH) peptides. (In these peptides Ac denotes N^α -acetylation of the N-terminus, -OH means a peptide with a free a-carboxyl group at the C-terminus and the‘ox’denotes the oxidation of the sulphhydryl groups of Cys–129 and Cys–142.) Both acetylated peptides were also able to inhibit the binding of βGalNAc(1–4)βGal-biotin to the corresponding BSA-Peptide(128–144)_ox-OH conjugates. The βGlcNAc(1–3)βGal(1–4)βGlc-biotin conjugate was unable to specifically bind to either Immobilized PAK and PAO pili or the respective C-termlnal peptides. The data above demonstrated that the P. aeruginosa pili recognize asialo-GM₁ receptor analogue and that βGalNAc(1–4)βGal disaccharlde is sufficient for binding. Furthermore, the binding to βGalNAc(1–4)βGal was mediated by residues 128–144 of the pilin subunit.     

The Influence of the Synthetic Growth Regulator α-Chloro-(β-(3-chloro-o-tolyl) propionitrile on Some Phytochrome-mediated Processes

In intact Sinapis alba seedlings the synthetic growth regulator α-chloro-β-(3-chloro-o-tolyl)propionitrile (PRB-8) inhibits the phytochrome-mediated anthocyanin synthesis, carotenoid synthesis and L-phenylalanine ammonia-lyase activity. This action was compared with that of other auxins and with that of α-chloro-β-(3-chloro-o-tolyl)propionitrile-homologues. From the results it appeared that PRB-8 and some other o-tolyl derivatives are more inhibitive than other known auxins.     

α-N-Acetyl β-Endorphins in the Pituitary: Immunohistochemical Localization Using Antibodies Raised Against Dynorphin(1-13)

Abstract: Intense immunohistochemical staining of the intermediate lobe of the pituitary was observed by using an antiserum raised against synthetic dynorphin(1-13) treated with a water-soluble carbodiimide (CDI). Subsequent studies showed that the immunostaining was blocked by preincubation of the antiserum with acetylated derivatives of both β-endorphin and dynorphin(1-13) as well as by CDI-treated dynorphin(1-13), but only weakly by authentic dynorphin(1-13). Neither nonacetylated β-endorphin nor any other fragments of the ACTH/endorphin precursor blocked the immunostaining of the intermediate lobe. Analysis of the CDI-treated dynorphin(1-13) used as an antigen showed that most of the peptide was acetylated at primary amino groups. CDI treatment of dynorphin(1-13) results in the formation of an acetyl derivative because the commercially available peptide is supplied as the acetate salt. The antibodies responsible for the intermediate lobe staining were isolated by affinity chromatography, using a column containing partially purified intermediate lobe extract linked to an affinity resin and a radioimmunoassay (RIA) was developed with CDI-treated dynorphin(1-13) used as a trace and as a standard. Competition studies showed 0.5-1% cross-reactivity with α-N-acetyl β-endorphin(1-31), α-N-acetyl β-endorphin(1-27), and totally acetylated β-endorphin(1-31). Nonacetylated β-endorphins did not cross-react. Posterior-intermediate lobe extracts from rat and beef were fractionated by gel filtration. Rat posterior-intermediate lobe extracts were also fractionated by cation-exchange chromatography. Fractionated extracts were analyzed by RIAs for β-endorphin, CDI-treated dynorphin(1-13), and authentic dynorphin(1-13). The results suggested that the peptides responsible for the intermediate lobe staining were mainly four different derivatives of β-endorphin bearing an acetyl group at the amino terminus. No immunostaining was seen in the posterior and anterior lobes of the pituitary. This suggests that the intermediate lobe is the main source of acetylated β-endorphins in the pituitary.     

DNA sequence of the porcine α-lactalbumin 5' flanking region and single-base polymorphisms within this region

The 5' flanking region of the α-lactalbumin (α-LA) gene was sequenced for the Duroc, Yorkshire and Meishan breeds of swine to identify potential sequence variants within this regulatory region of the porcine α-LA gene. The sequenced region of the gene encompasses 391 bp 5' of the translation start site to 11 bp 3' of the translation start site. Within this sequence of the porcine α-LA gene two single-base pair differences were detected. One variant occurs at position – 178 and the other at position – 235 from the translation start site. Each of the variations can be detected by a restriction fragment length polymorphism within a polymerase chain reaction amplified product. The polymorphisms at the – 178 and – 235 positions appear to be genetically linked in the animals that have been analysed.     

Isolation of genes encoding β-D-xylanase, β-D-xylosidase and α-L-arabinofuranosidase activities from the rumen bacterium Prevotella ruminicola B14

Abstract Prevotella ruminicola B₁4 is a strictly anaerobic, Gram-negative, polysaccharide-degrading rumen bacterium. Xylanase activity in this strain was found to be inducible, the specific activity of cells grown on xylan being increased at least 20-fold by comparison with cells grown on glucose. Ten bacteriophage clones expressing xylanase activity were isolated from a A EMBL3 genomic DNA library of P. ruminicola B₁4. These clones were shown to represent four distinct chromosomal regions, based on restriction enzyme analysis and DNA hybridisation. Three groups of clones encoded activity against oat spelt xylan but not carboxymethylcellulose (CMC). In one of these groups, represented by clone 5, activities against pNP-arabinofuranoside and pNP-xyloside were found to be encoded separately from endoxylanase activity. The fourth region encoded activity against CM cellulose and lichenan, in addition to xylan, and contains an endoglucanase/xylanase gene isolated previously.     

The α5(H105R) mutation impairs α5 selective binding properties by altered positioning of the α5 subunit in GABAA receptors containing two distinct types of α subunits

GABA_A receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA_A receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABA_A receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.