Ion conductivity through TEMPO-mediated oxidated and periodate oxidated cellulose membranes.

Cellulose in different forms is increasingly used due to sustainability aspects. Even though cellulose itself is an isolating material, it might affect ion transport in electronic applications. This effect is important to understand for instance in the design of cellulose-based supercapacitors. To test the ion conductivity through membranes made from cellulose nanofibril (CNF) materials, different electrolytes chosen with respect to the Hofmeister series were studied. The CNF samples were oxidised to three different surface charge levels via 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), and a second batch was further cross-linked by periodate oxidation to increase wet strength and stability. The outcome showed that the CNF pre-treatment and choice of electrolyte are both crucial to the ion conductivity through the membranes. Significant specific ion effects were observed for the TEMPO-oxidised CNF. Periodate oxidated CNF showed low ion conductivity for all electrolytes tested due to an inhibited swelling caused by the crosslinking reaction.

Microrheology of novel cellulose stabilized oil-in-water emulsions.

Diffusing wave spectroscopy (DWS) is a powerful optical technique suitable to investigate turbid samples in a nondestructive and reproducible way, providing information on the static and dynamic properties of the system. This includes the relative displacement of emulsion droplets over time and changes in the viscoelastic properties. Here, novel and promising cellulose-based oil-in-water (O/W) emulsions were prepared and studied, for the first time, by DWS. Cellulose plays the role of a novel eco-friendly emulsifying agent. The hydrolysis time of cellulose was observed to affect the average size of the emulsion droplets and their stability; the longer the hydrolysis time, the more dispersed and stable the emulsions were found to be. Additionally, a good complementarity between the microrheology (DWS) and macrorheology (mechanical rheometer) data was found. Our work suggests that DWS is a highly attractive method to investigate the stability, aging and microrheology properties of cellulose-based emulsions, providing valuable insights on their microstructure. This technique is thus highly appealing for the characterization and design of novel emulsion formulations.

An experimental model for acute poststreptococcal glomerulonephritis in mice.

A number of factors have been implicated in the pathogenesis of acute poststreptococcal glomerulonephritis (APSGN). The lack of a reliable animal model has made it difficult to further examine the role of these factors in the pathogenetic process. In this report, we present a tissue cage model in mice for the study of APSGN. Morphological and immunohistological changes in the kidney, resembling those of APSGN in man, were induced at high frequency in the experimental model after infection with group A streptococcal nephritis isolates. Nephritis-associated strain induced hypercellularity, occlusion of capillaries, and C3 deposition at high frequencies compared to the changes induced in animals infected with a non-nephritis-associated strain and non-infected controls. In animals infected with a nephritis isolate, hematuria and proteinuria were also detected. If penicillin treatment was initiated on the third day of infection, the development of the nephritis process was prevented. Streptokinase, as well as preabsorbing antigen and streptococcal pyrogenic exotoxin B (SpeB), have been implicated in the pathogenesis of APSGN. These proteins, as well as SpeA and SpeF, were detected in the fluids of the infectious focus, regardless of the origin of the strains and whether or not glomerulonephritis was seen. Antibodies to streptokinase were evoked in the majority of the infected animals. This immune response did not correlate with the nephritic process since hypercellularity was also seen in animals which lacked detectable streptokinase antibodies. The results show that the mouse tissue cage model can be used to study APSGN and to evaluate factors involved in the pathogenesis of the disease.

The superantigenic activity of streptococcal pyrogenic exotoxin B is independent of the protease activity.

The nature of the mitogenic activity of pyrogenic streptococcal exotoxin B, also known as streptococcal cysteine protease, has been debated in the literature. Streptococcal exotoxin B has been shown to cleave interleukin-1beta precursor and create biologically active interleukin-1beta, a major cytokine mediating inflammation and shock. This activity could mimic the mitogenicity and cytokine release induced by superantigens in lymphocyte stimulating experiments. In this study, the protease activity of streptococcal exotoxin B was irreversibly inhibited by covalent binding of a tripeptide and the superantigenic properties of streptococcal exotoxin B were found not to be influenced by this inactivation. Native as well as protease-inactivated streptococcal exotoxin B was shown to stimulate T-cell proliferation without a need of metabolically active antigen presenting cells. Furthermore, streptococcal exotoxin B-induced T-cell proliferation was shown to require HLA-DQ since addition of HLA-DQ monoclonal antibodies totally inhibited the mitogenic activity of streptococcal exotoxin B, indicating that streptococcal exotoxin B, as other superantigens, makes direct contact with the T-cell receptor via HLA class II. The aim of this study was to characterize the relationship between the proteolytic and superantigenic properties of streptococcal exotoxin B.

Identification of two genes, cpsX and cpsY, with putative regulatory function on capsule expression in group B streptococci.

Two divergently transcribed open reading frames: cpsX and cpsY separated by a common regulatory region was identified upstream of the cpsA-D genes involved in polysaccharide capsule biosynthesis in group B streptococci (GBS). We suggest that these genes are involved in the regulation of capsule expression in GBS, since the CpsX protein shares sequence similarities with LytR of Bacillus subtilis, an attenuator of transcription while CpsY has similarity to a wide variety of members of the LysR family of transcriptional regulators. No deletions, insertions, DNA rearrangements, or apparent differences were discovered in the postulated regulatory genes when the gene region was compared in GBS with different capsule phenotypes. Thus, other yet unidentified gene loci may control capsule phase variation in GBS.

Magnetostatic image current and its application to an analytic identification of a current dipole inside a conducting sphere.

The image solution for the static magnetic field outside a conducting sphere with an internal current dipole is considered. The image current, which is a linear distribution of magnetic dipoles on the line segment between the dipole point and the center of the sphere, is derived by using the fact that the induced current does not have any contribution to the radial component of the magnetic field outside the sphere. The image is then used to obtain some explicit formulas for identifying the location and tangential moment of the primary current dipole. This explicit identification method is also tested with a real model for a patient's brain.

The presence of conjugative transposon Tn916 in the recipient strain does not impede transfer of a second copy of the element.

Transfer of the conjugative transposon Tn916 from the chromosome of Bacillus subtilis to a transposon-free Streptococcus pyogenes strain occurs at the same frequency as transfer to a Tn916-containing recipient. This rules out a model for conjugal transfer of Tn916 in which a copy of the element in the recipient represses transposition of a copy introduced by conjugation. Homology-directed integration of the incoming transposon into the resident one is less frequent than insertion elsewhere in the chromosome. This shows that after conjugation, transposition occurs more frequently than homologous recombination. However, because transconjugants arising from homologous recombination can be selected, it is possible to use Tn916 as a shuttle for gram-positive organisms for which there is no easy means of introducing DNA.

Biogenesis of E. coli Pap pili: papH, a minor pilin subunit involved in cell anchoring and length modulation.

The biogenesis of Escherichia coli Pap pili, encoded by the pap gene cluster, was studied. A novel gene, papH, was identified and found to encode a weakly expressed pilin-like protein. PapH was dispensable for digalactoside-specific binding and for formation of Pap pili. However, in papH deletion mutants 50%-70% of total pilus antigen was found free of the cells. We present evidence showing coregulation of papH and the adjacent gene, papA, which encodes the major pilin subunit. A decrease in the PapA to PapH ratio resulted in a large fraction of cells producing shortened pili, whereas overproduction of PapA relative to PapH resulted in cells with lengthened pili. The data show that PapH has roles in anchoring the pilus to the cell and in modulating pilus length.

Streptokinase activity among group A streptococci in relation to streptokinase genotype, plasminogen binding, and disease manifestations.

Certain genotypic variants of streptokinase (ska) of beta-hemolytic streptococci group A have been associated with acute post-streptococcal glomerulonephritis (APSGN). In our earlier studies on strains isolated from Ethiopian children with various streptococcal disease manifestation, we reported an even distribution of streptokinase genotypes with no association to disease patterns. Considering the possibility that strains could differ in their ability to secrete the protein, levels of streptokinase activity in culture supernatants of these strains were determined by a plasminogen activation assay using a synthetic tripeptide, H-D-valyl-leucyl-lysin-p-nitroaniline, as a substrate. Of the 53 streptococcal group A strains, ten (19%), which belonged to genotype ska4 and ska8, did not activate human plasminogen. These strains did not activate bovine, sheep, horse, rabbit or porcine plasminogens either. They represented at least five M protein and non-typeable serotypes, and were characterized by high human plasminogen binding activity. Six of the 53 strains (11%) harbouring genotype ska3 and ska7 showed low levels of human plasminogen activation. Strains of ska1 and ska2, 37/53, activated human plasminogen at a higher level (p < 0.005). Levels of plasminogen activation were not significantly different among the ska1 and ska2 strains associated with various streptococcal disease manifestations. Antibody levels against streptokinase were higher (p < 0.05) in convalescent sera from acute rheumatic fever and APSGN patients in comparison with sera from other patient categories and healthy controls. Streptokinase genotype and in vitro streptokinase production do not correlate directly to streptococcal disease manifestation, indicating a probable significance of additional streptococcal and/or host factors in the initiation of APSGN.

Phase-shift of polysaccharide capsule expression in group B streptococci, type III.

The type-specific polysaccharide capsule is an important virulence determinant in group B streptococci (GBS). The previously described inverse relationship between the buoyant density of a GBS-isolate and the capsular thickness was used to assess the frequencies of polysaccharide capsular phase-shift in clinical GBS, type III strains. Shift from intermediate density (ID) of parental strains, to high density (HD), i.e. shift from intermediate capsule thickness to poor encapsulation, was found to range from 1.2 x 10(-3) to 4.8 x 10(-6). Shift from ID to low density (LD), i.e. shift to abundant encapsulation, ranged from 1.9 x 10(-4) to 1.1 x 10(-7). Shifts were reversible in all cases, either directly (HD-->LD or vice versa) or through intermediate forms. Reversion frequencies were in some isolates as high as 10(-1). Phase-shift frequencies differed more than a thousand-fold between compared strains. Differences in phenotypic shift between strains were validated using flow cytometry. Possible modulation of capsule expression by changes in culture conditions was assessed. Variation of temperature, oxygen-tension, and presence of human serum did not affect capsule expression. However, growth at pH below 5.5 decreased the amount of capsule bound native type III polysaccharide, probably through phenotypic modification rather than genetic shift. IS861, an insertion sequence which has been proposed a regulatory function on the GBS capsule expression, was found in multiple copies in the isolates investigated. No differences in copy number or location of IS861 between the differently encapsulated phenotypes were found.

Mutually exclusive distribution of IS1548 and GBSi1, an active group II intron identified in human isolates of group B streptococci.

The present study shows that active, self-splicing group II intron GBSi1 is located downstream of the C5a-peptidase gene, scpB, in some group B streptococcus (GBS) isolates that lack insertion sequence IS1548. IS1548 was previously reported to be often present at the scpB locus in GBS isolated in association with endocarditis. Since none of 67 GBS isolates examined, 40 of which were of serotype III, harbored both IS1548 and GBSi1, these two elements are suggested to be markers for different genetic lineages in GBS serotype III. The DNA region downstream of scpB in GBS isolates harboring either GBSi1, IS1548, or none of these mobile elements was found to encode the laminin binding protein, Lmb, which shows sequence similarities to a family of streptococcal adhesins. IS1548 is inserted 9 bp upstream of the putative promoter for lmb, while the insertion site for GBSi1 is located 88 bp further upstream. Sequences highly similar to GBSi1 exist also in Streptococcus pneumoniae. An inverted repeat sequence, with features typical of transcription terminators, was identified immediately upstream of the insertion site for the group II intron both in the GBS and S. pneumoniae sequences. This motif is suggested to constitute a target for the GBS intron as well as for rather closely related introns in Bacillus halodurans, Pseudomonas alcaligenes, and Pseudomonas putida. When transcripts containing the GBSi1 intron were incubated at high concentrations of ammonium and magnesium, a major product with the expected length and sequence for the ligated exons was generated. Unlike, however, all members of group II investigated so far, the excised intron was in linear, rather than in a branched (lariat), form.

Streptococcal DNase B is immunologically identical to superantigen SpeF but involves separate domains.

The previous suggestion that streptococcal superantigen SpeF might be identical to DNase B was confirmed in this study. Polyclonal SpeF-specific antisera were able to inhibit depolymerization of methyl-green DNA by DNase B. However, T-cell mitogenicity and nuclease activity appear to involve separate immune epitopes on SpeF, since sera with the capacity to neutralize the mitogenic activity of SpeF did not always inhibit the DNase activity.

Characterization and nucleotide sequence of a Klebsiella oxytoca cryptic plasmid encoding a CMY-type beta-lactamase: confirmation that the plasmid-mediated cephamycinase originated from the Citrobacter freundii AmpC beta-lactamase.

Plasmid pTKH11, originally obtained by electroporation of a Klebsiella oxytoca plasmid preparation into Escherichia coli XAC, expressed a high level of an AmpC-like beta-lactamase. The enzyme, designated CMY-5, conferred resistance to extended-spectrum beta-lactams in E. coli; nevertheless, the phenotype was cryptic in the K. oxytoca donor. Determination of the complete nucleotide sequence of pTKH11 revealed that the 8,193-bp plasmid encoded seven open reading frames, including that for the CMY-5 beta-lactamase (blaCMY-5). The blaCMY-5 product was similar to the plasmidic CMY-2 beta-lactamase of K. pneumoniae and the chromosomal AmpC of Citrobacter freundii, with 99.7 and 97.0% identities, respectively; there was a substitution of phenylalanine in CMY-5 for isoleucine 105 in CMY-2. blaCMY-5 was followed by the Blc and SugE genes of C. freundii, and this cluster exhibited a genetic organization identical to that of the ampC region on the chromosome of C. freundii; these results confirmed that C. freundii AmpC was the evolutionary origin of the plasmidic cephamycinases. In the K. oxytoca host, the copy number of pTKH11 was very low and the plasmid coexisted with plasmid pNBL63. Analysis of the replication regions of the two plasmids revealed 97% sequence similarity in the RNA I transcripts; this result implied that the two plasmids might be incompatible. Incompatibility of the two plasmids might explain the cryptic phenotype of blaCMY-5 in K. oxytoca through an exclusion effect on pTKH11 by resident plasmid pNBL63.

A method for allelic replacement that uses the conjugative transposon Tn916: deletion of the emm6.1 allele in Streptococcus pyogenes JRS4.

The emm6.1 allele of Streptococcus pyogenes JRS4 was deleted by using the conjugative transposon Tn916. The aphA-3 gene, conferring resistance to kanamycin, was cloned between the sequences flanking the structural gene for the type 6 M protein (emm6.1) and inserted into the BstXI site of Tn916 to generate the chimeric transposon Tn916-5K3. Because the BstXI site lies in a nonessential region of Tn916, the chimeric transposon could transfer by conjugation from Bacillus subtilis into JRS4. In some of the transconjugants, Tn916-5K3 replaced the emm6.1 locus of JRS4 by homologous recombination between the cloned emm6.1-flanking regions and the resident chromosome. One recombinant studied in detail, JRS75, was kanamycin resistant and tetracycline sensitive and lacked immunologically detectable M6 protein. Furthermore, by Southern blot analysis, the DNA region encompassing the emm6.1 structural gene was found to have been replaced by aphA-3.

Adhesion to human cells by Escherichia coli lacking the major subunit of a digalactoside-specific pilus-adhesin.

Pathogenic bacteria frequently possess pili with specific binding properties that allow them to attach to epithelial tissue. In Escherichia coli, the pili associated with pyelonephritis (Pap pili) bind to digalactoside-containing glycolipids on the uroepithelium. Transposon-insertion mutants and deletion mutants of the cloned genetic determinant encoding synthesis of such digalactoside-binding Pap pili have been studied in E. coli K-12. Mutants that completely lack synthesis of the major Pap pili subunit protein, the papA gene product, and thereby no longer produce pili were shown to retain the binding specificity of intact Pap pili. Reduced expression of some of the remaining pap genes, presumably due to polarity effects from papA::Tn5 insertions, was circumvented by the use of a copy-number mutant plasmid vector. Derivatives carrying the papA-D genes produced Pap pili but did not bind to human cells. The products of the genes papE-G are essential for digalactoside-specific hemagglutination and for attachment to urinary bladder cells. The papC and papD genes presumably aid in surface localization and/or polymerization of the pili-adhesin subunits and are required for expression of pili as well as of the binding properties. Serological evidence is presented that suggests that a minor pilus component(s), presumably produced by the papE, -F, or -G gene, is the actual binding moiety in the digalactoside-specific interaction of Pap pilus-adhesin.

Nucleotide sequence, regulation and functional analysis of the papC gene required for cell surface localization of Pap pili of uropathogenic Escherichia coli.

The papC gene of uropathogenic Escherichia coli is required for the formation of digalactoside-binding Pap pili. papC forms part of an operon wherein the regulatory gene papB, the major pilin gene papA, a minor pilin-like gene papH, and papC are co-transcribed. Furthermore, the extent of PapC synthesis was found to affect the number of pili expressed on the cell surface. The DNA sequence of the papC gene is presented and its deduced amino acid sequence is compared to that of the FaeD protein encoded by the K88 pili gene cluster. The PapC protein was localized to the E. coli outer membrane where it may form a trans-membrane channel through which pilin subunits are surface localized.

Endocarditis caused by a group B Streptococcus strain, type III, in a nonencapsulated phase.

A nontypeable blood isolate of group B streptococci (GBS) from a patient with endocarditis is suggested to be the nonencapsulated phase of a GBS strain, type III. From the original high-density isolate, a low-density, encapsulated phase was selected by Percoll gradient centrifugation. This phenomenon should be considered before a GBS strain is classified as truly nontypeable.

Genetic diversity in T1M1 group A streptococci in relation to clinical outcome of infection.

Genetic diversity was found at high frequency downstream of the emm1 gene among T1M1 group A streptococci (GAS) isolated in Scandinavia during a recent epidemic. Clonal variation was also seen in the speA and speB genes but at much lower frequency; no variation was detected in the speC gene. Erythrogenic toxin A was found to be expressed at low levels in all strains; erythrogenic toxins B and C were produced in high amounts. All strains were found to harbor the speA, speB, and speC genes, regardless of the amount of toxin produced. No correlation was found between one specific T1M1 clone and the more serious infections when isolates from bacteremic patients (fatalities or survivors), those with uncomplicated infections, and healthy carriers were compared. Similar results were obtained in a family study in which 3 family members were found to be asymptomatic carriers of the same GAS T1M1 clone as in the bacteremic patient, defined by genotypic and phenotypic experiments.