Nitrogen saturation induced during winter by experimental NH4NO3 addition to a forested catchment.

To investigate the potential risk of 'nitrogen saturation' in Scandinavian boreal forests, the authors are experimentally adding 30-50 kg N ha(-1) year(-1) as NH4NO3 in precipitation to an entire 0.52-ha 80-year-old spruce forested catchment at Gårdsjön, near Gothenburg on the Swedish west coast. NO3 concentrations in runoff increased from 0 to about 7 microeq liter(-1) (maximum pulse of 43 microeq liter(-1)). The increase occurred in winter; during the April-October growing season, NO3 concentrations were very low. The speed of the response suggests that these forests are already close to saturation.

Sulphur and seasalt deposition as reflected by throughfall and runoff chemistry in forested catchments.

At forested catchments at Lake Gårdsjön on the Swedish west coast the deposition and runoff chemistry has been followed during the period 1979-1990 by throughfall and runoff measurements as well as by measurements of atmospheric concentrations. The 10-year means in throughfall and runoff are very similar for sulphur and the main seasalt ions sodium and chloride; for sulphur 26.1 and 27.6 kg ha(-1) yr(-1), for sodium 49 and 52 kg ha(-1) yr(-1) and for chloride 96 kg ha(-1) yr(-1) and 93 kg ha(-1) yr(-1), respectively. The actual flows are 100-200% higher than the wet deposition as collected in open bulk precipitation collectors indicating a very large input by dry deposition. One important question is to what extent the throughfall and runoff values can be used as measures of total deposition. We present results from studies at different experimental catchments illustrating the possibilities of using throughfall and runoff data as measures of atmospheric deposition of sulphur and seasalt.

Use of synthetic oligodeoxyribonucleotides for type-specific identification of coxsackie B viruses.

Synthetic oligodeoxyribonucleotides were used for type-specific identification of members of the coxsackie B virus group by nucleic acid hybridization. Two pairs of oligonucleotide chains were constructed based on nucleotide sequences in the VP1 regions of coxsackieviruses B3 and B4. Each labelled probe had a length of 24 nucleotides. The results showed that the oligonucleotide hybridized in a type-specific manner when assayed with extracts from cells infected with all different coxsackie B viruses. A method based on similar principles may thus be used for enterovirus typing.

Expression of human insulin-like growth factor I in bacteria: use of optimized gene fusion vectors to facilitate protein purification.

Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.

A novel adenovirus-2 E1A mRNA encoding a protein with transcription activation properties.

Two novel adenovirus-2 early region 1A mRNAs, designated 10S and 11S, have been characterized. They differ from the previously described 9S, 12S and 13S mRNAs by having an additional intron removed during mRNA maturation. The 10S and 11S mRNAs encode proteins with mol. wts of 30 and 35 kd. These proteins are encoded in the same translational reading frame as the 12S and 13S mRNA products and differ by lacking 72 amino acids between position 27 and 98. A functional analysis showed that both the 10S and 11S mRNA products are non-essential for lytic virus growth, and, furthermore, defective in cellular transformation. Interestingly the 11S mRNA product functioned as an efficient transcriptional activator in transient expression assays but was very ineffective as a gene activator during virus growth. Moreover, the virus expressing the 11S cDNA failed to block host cell gene expression although substantial amounts of late proteins were expressed. From the biological properties of the E1A cDNA mutants it was possible to localize two functional domains in the E1A proteins; one region required for transcriptional activation (amino acids 140-185), and a second domain required for adenovirus transformation and the control of viral and cellular gene expression during a lytic infection (amino acids 27-98).

P-fimbriae of pyelonephritogenic Escherichia coli: identification and chemical characterization of receptors.

Pyelonephritogenic Escherichia coli isolates were found to be equal in their ability to adhere to uroepithelial cells and to cause agglutination specific for human erythrocytes. This adhesive capacity was not affected by D-mannose and was found to be mediated by a new class of E. coli fimbriae named P-fimbriae. Using human erythrocytes of different blood groups, we found the receptor molecules for P-fimbriae to be associated with the P blood group antigens, i. e. glycosphingolipids corresponding to the P blood group antigens P, P1 and Pk. By agglutination studies of erythrocytes with various set-ups of these antigens as well as by inhibition studies using synthetic saccharide derivatives and by coating non-agglutinable erythrocytes with a synthetic glycolipid, the minimal receptor structure was identified as the alpha-D-Galp-(1-4)-beta-D-Galp moiety of the carbohydrate portion of these glycosphingolipids. Similar experiments on isolated uroepithelial cells proved that these glycosphingolipids also constitute the receptors in the urinary tract.

P-fimbriae of pyelonephritogenic Escherichia coli: significance for reflux and renal scarring-a hypothesis.

An experimental pyelonephritis model was developed in monkeys (Macaca fascicularis) using P-fimbriated Escherichia coli as the infecting organism. The relevant receptor molecules for P-fimbriae were also shown to be present in Macaca fascicularis. Atraumatic administration of P-fimbriated E. coli into the ureter induced a ureteritis followed by acute and chronic pyelonephritis. The decisive role of P-fimbriae as an adhesive virulence factor was proven by the receptor blockade of P-fimbriae-mediated bacterial adhesion by a synthetic receptor analogue (alpha-D-Galp-(1-4)-beta-D-Galp-1-OMe), which was administered into the ureter together with the challenge bacteria. On the basis of these and other findings, the role of reflux and pyelonephritis in relation to renal scarring is discussed in this paper. It is proposed that minor transitional vesicoureteral reflux together with the adhesive property of P-fimbriated E. coli and their ability to induce ureteritis might constitute an alternative mechanism to gross reflux by which bacteria ascend to the kidney. These findings and the fact that intestinal colonization with P-fimbriated E. coli coincides with the disease have opened up new prophylactic and therapeutic possibilities.

Synthesis of di- and tri-saccharides corresponding to receptor structures recognised by pyelonephritogenic E. coli fimbriae (pili).

Syntheses are described of di- and tri-saccharides required for studies of the inhibition of adhesion by means of fimbriae of pyelonephritogenic E. coli bacteria to epithelium cells in the urinary tract containing suitable receptor sites. The disaccharides are the rho-nitrophenyl and methyl glycosides of 4-omicron-alpha-D-galactopyranosyl-beta-D-galactopyranose, and the trisaccharides are the rho-nitrophenyl and methyl glycosides of 4-omicron-(4-omicron-alpha-D-galactopyranosyl-beta-D-galactopyranosyl)-beta- D-glucopyranose. The key aglycons were the 1,2,3,6-tetrabenzoate of alpha-D-galactose and the 1,2,3,6,-2',3',6'-heptabenzoate of alpha-lactose. Glycosidations were performed with 2,3,4,6-tetra-omicron-benzyl-alpha-D-galactopyranosyl chloride as glycosylating agent and silver triflate as promoter.

P-antigen-recognizing fimbriae from human uropathogenic Escherichia coli strains.

P-antigen-recognizing fimbriae (P fimbriae) from four pyelonephritogenic Escherichia coli strains and type 1 fimbriae from an E. coli strain and a Salmonella typhimurium strain were purified. The P fimbriae were morphologically similar to type 1 fimbriae. The purified P fimbriae agglutinated neuraminidase-treated human P1 and P2k erythrocytes but not p erythrocytes, which lack all P-blood group-specific glycosphingolipids. However, coating of neuraminidase-treated p erythrocytes with globoside rendered such erythrocytes agglutinable by the P fimbriae. The hemagglutinations were in all instances fully inhibited by the synthetic alpha-D-Galp-(1-4)-beta-D-Galp-1-O-Me glycoside. The binding specificity of the P fimbriae could also be demonstrated by using fimbriae coated onto latex particles and nontreated erythrocytes. It was thus concluded that the P fimbriae recognize and bind to the alpha-D-Galp-(1-4)-beta-D-Galp carbohydrate sequence occurring in the series of P-blood group antigen-specific glycosphingolipids. In contrast to both type 1 fimbriae, all four P fimbriae preparations showed multiple bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antisera were raised in rabbits against the various E. coli fimbriae. In enzyme-linked immunosorbent assays each one of the antisera to the P fimbriae reacted to titers of log 4 to 7 with both the homologous and the heterologous P fimbriae, but not with the type 1 fimbriae of E. coli. In a reciprocal fashion, the antiserum to the type 1 fimbriae of one E. coli strain reacted only with the homologous type 1 but not with any of the P fimbriae preparations.

Rapid identification of P-fimbriated Escherichia coli by a receptor-specific particle agglutination test.

Most (greater than 90%) Escherichia coli strains isolated from children with acute non-obstructive pyelonephritis exhibit a specific type of filamentous protein appendage known as P-fimbriae. These fimbriae enable the bacterium to adhere to human uroepithelial cells by the specific recognition of and binding to a particular class of glycosphingolipids correlated to the human P-blood group antigens. In this paper a new method for the rapid and reliable identification of such P-fimbriated pyelonephritogenic bacteria is described. The method is based on particles to which the minimal glycoside receptor structure recognized by P-fimbriae is attached. Mixing these receptor-containing particles with P-fimbriated bacteria results in a strong and immediate agglutination reaction. The specificity and sensitivity of this new particle agglutination test proved to be superior to the haemagglutination assay previously used.

Mannose-resistant haemagglutination and P antigen recognition are characteristic of Escherichia coli causing primary pyelonephritis.

Thirty-two Escherichia coli strains from 30 children with pyelonephritis were examined for their haemagglutination patterns and O and K serotypes. 29 (91%) of the strains showed mannose-resistant haemagglutination (MRHA). By use of well-defined target cells, these MRHA+ strains could be shown to recognise human cells either in a P-specific manner (recognising a specific galactosyl-galactose structure which is part of P blood groups antigens) or in a separate, X-specific manner. Both recognition mechanisms could occur separately or together on the same bacteria, the frequencies of P and X specificity being 81 and 19%, respectively. Both MRHA and P specificity were significantly associated with the O antigens 01, 04, 06, 016, and 018, and the capsular antigen K1, which have previously been associated with pyelonephritis. However, the association of MRHA and P specificity with upper urinary tract infection in children is greater than that of any other laboratory-defined bacterial characteristic.

Occurrence of P-fimbriated Escherichia coli in urinary tract infections.

The occurrence of Escherichia coli possessing P blood-group-specific adhesins (P-fimbriae) was examined in 97 children with urinary tract infections and 82 healthy controls. P-fimbriae were present in 91% (33/35) of the urinary strains causing acute pyelonephritis. Among strains causing cystitis and asymptomatic bacteriuria P-fimbriae were found in 19% and 14% of cases, respectively. Only 7% of faecal isolates from healthy controls carried P-fimbriae. The results were similar in three different studies. In most of the children with acute pyelonephritis the urinary pathogen was the predominant E. coli strain of the periurethral and faecal flora.

Structure of carbohydrate part of receptor on human uroepithelial cells for pyelonephritogenic Escherichia coli.

The binding of pyelonephritogenic Escherichia coli strains to human uroepithelial cells from patients with and without P blood group antigens was investigated. Uroepithelial cells from p phenotypes bound pyelonephritogenic e. coli to a significantly lesser extent than did cells from P1 and P2 phenotypes. The binding of pyelonephritogenic E. coli to urinary epithelial cells of P1 phenotypes was blocked by the synthetic disaccharide alpha-D-Galp-(1 leads to 4)-beta-D-Galp whose structures is related to that of the P blood group antigens. Coating of P1 cells with a synthetic disaccharide derivative increased the binding of bacteria. None of 30 individuals of p phenotype had had urinary tract infection. The findings show that the disaccharide alpha-D-Galp-(1 leads to 4)-beta-D-Galp, previously shown to be the erythrocyte receptor for the fimbriae of pyelonephritongenic E. coli, is also the receptor structure on uroepithelial cells.

Identification of a carbohydrate receptor recognized by uropathogenic Escherichia coli.

Earlier investigations have shown that pyelonephritic Escherichia coli specifically recognize and bind to carbohydrate structures correlated to the P blood group antigens. These findings are confirmed and extended in this study. Twenty-two of 23 nonselected E. coli strains from children with acute febrile pyelonephritis failed to agglutinate human erythrocytes lacking the antigens within the P blood group system. Only one of 32 faecal isolates exhibited this specific agglutinating property. The new informatin in this paper is that P2k erythrocytes, containing only the Pk antigen, were agglutinated to the same extent by pyelonephritic E. coli strains, giving further support to the proposal that the Pk glycosphingolipid is related to the receptor for pyelonephritic E. coli. In addition, the importance of the oligosaccharide moiety of the Pk glycosphingolipid for the binding of E. coli was further investigated. The synthesized disaccharide alpha-D-Galp-(1-4)-beta-D-Galp-1-O-0-NO2 inhibited the agglutination of human erythrocytes caused by two pyelonephritic E. coli strains at concentrations of less than 1 mM. Hence, the minimal receptor structure recognized by these E. coli strains appears to be the alpha-D-Galp-(1-4)-beta-D-Galp structure. How generally valid this observation may be needs further investigation. The findings may open new possibilities for diagnosis and treatment of urinary tract infection.