Hydrogen peroxide is a true first messenger.

Hydrogen peroxide has been shown to act as a second messenger mediating intracellular redox-sensitive signal transduction. Here we show that hydrogen peroxide is also able to transmit pro-inflammatory signals from one cell to the other and that this action can be inhibited by extracellularly added catalase. If these data can be further substantiated, hydrogen peroxide might become as important as nitric oxide as a small molecule intercellular (first) messenger.

Structural and functional characterization of B-domain deleted recombinant factor VIII.

A new high-purity recombinant factor VIII preparation has been developed for the treatment of hemophilia A. Structurally, this factor VIII preparation, B-domain deleted recombinant factor VIII (BDDrFVIII), differs from other recombinant and plasma-derived factor VIII preparations in that most of the B-domain has been deleted. To ensure that BDDrFVIII contains the requisite structural and functional features, it has been subjected to detailed biochemical and biophysical characterization in comparison to the plasma-derived form of factor VIII. Laboratory studies have shown that the primary, secondary, and tertiary structures of BDDrFVIII and the posttranslational modifications are similar to those of the [80 + 90]-kd form of plasma-derived factor VIII. In addition, BDDrFVIII has full biologic activity compared with full-length factor VIII preparations.

Structural and functional characteristics of the B-domain-deleted recombinant factor VIII protein, r-VIII SQ.

Recombinant factor VIII SQ (r-VIII SQ), ReFacto, is a recombinant factor VIII product similar to the smallest active factor VIII protein found in plasma-derived factor VIII (p-VIII) concentrates. The protein comprises two polypeptide chains of 80 and 90 kDa and lacks the major part of the heavily glycosylated B-domain i.e. amino acids Gln744 to Ser1637. r-VIII SQ retains six potential glycosylation sites for N-linked oligosaccharides at asparagine residues 41, 239, 582, 1685, 1810 and 2118. We describe a thorough comparison of the characteristics of r-VIII SQ with those of p-VIII. The primary and secondary structures of r-VIII SQ were in good agreement with that of B-domain-deleted p-VIII (p-VIII-LMW) as shown by SDS-PAGE, Western blotting with antifactor VIII antibodies, tryptic mapping, amino acid sequence analysis and circular dichroism spectroscopy. A few divergences also existed. Thus r-VIII SQ was shown to contain a small amount of the single chain primary translation product of 170 kDa and also the product specific sequence of 14 amino acids, the SQ-link, in the C-terminal end of the 90 kDa chain. It was shown that r-VIII SQ had a high specific activity of about 14,000 IU VIII:C/mg as determined by use of a chromogenic substrate assay. The r-VIII SQ protein was comparable to p-VIII forms with a retained B-domain, in terms of potency measured by a chromogenic substrate or a two-stage clotting assay, in interactions with thrombin, and with activated protein C (APC) in combination with Protein S. The ability of r-VIII SQ to participate as a cofactor in factor Xa generation in a mixture of factors IXa and X, phospholipid and calcium was in conformity with that of p-VIII. Furthermore r-VIII SQ had a good binding capacity for phospholipid vesicles and von Willebrand factor (vWF) as shown in gel filtration studies. The same kinetics in binding to von Willebrand factor was found for r-VIII SQ and p-VIII as determined by real-time biospecific interaction analysis (BIA) with use of the BIAcore instrument. The apparent association rate constant was 4 x 10(6) M(-1)s(-1). Two dissociation rate constants were found, 1 X 10(-2)s(-1) and 4 x 10(-4)s(-1). The results extend the present knowledge that the factor VIII B-domain is dispensable for the factor VIII cofactor function in hemostasis.

Airborne birch and grass pollen allergens in street-level shops.

Polluted urban outdoor air may be enriched with large amounts of submicronic respirable pollen allergen particles that penetrate into street-level shops. The objectives of the study were to map concentrations of birch and grass pollen allergens in indoor air of street-level shops and to explore the effect of electrostatic air cleaning under authentic working conditions, indoor air samples were collected in May and July 1999 in two shops. Allergens were quantified by a direct on sampling filter in solution (DOSIS) luminescence immunoassay. The average concentration of airborne indoor birch pollen allergen in the shop with air cleaning was estimated to be 20 +/- 9 SQ/m3 (mean +/- SD) compared to 31 +/- 17 SQ/m3 (mean +/- SD) of that without. The air cleaner reduced the indoor air birch pollen allergen concentration by on average 26 to 48% (P < 0.05). Corresponding figures for airborne indoor grass pollen allergen concentrations were 14 +/- 7 SQ/m3 and 17 +/- 8 SQ/m3, indicating a statistically non-significant (t-test) average 18% reduction of allergen by air cleaning. Excluding two observations with poor fit to the statistical model a significant (P < 0.05) average 27% reduction was obtained. Substantial amounts of airborne birch and grass pollen allergens may occur in street-level shops during flowering seasons.

Inflammatory bowel disease in children and adolescents in Sweden, 1984-1995.

BACKGROUND: A prospective study of inflammatory bowel disease (IBD) in Sweden was performed to investigate whether the incidence and morbidity have changed from 1984 through 1995. METHODS: Children 15 years of age or less with IBD were included--i.e., those with a definite diagnosis of ulcerative colitis (UC) and Crohn's disease (CD) and those classified as having indeterminate colitis (IC) and probable Crohn's disease (PCD). The study covered 56.5% of the pediatric population of Sweden. RESULTS: The diagnosis of IBD was made in 639 children, which corresponds to a mean annual incidence of 5.8 per 100,000. The incidence increased from 4.6 per 100,000 per year from 1984 through 1986 to 7.0 from 1993 through 1995. It reflected an increase in UC from 1.4 to 3.2 per 100,000 per year, which is a significant yearly percentage of increase (8%; confidence interval, 2-14%; P < 0.05). In contrast, no change occurred in the incidence of CD (1.2-1.3 per 100,000). The incidence of IC and PCD also remained fairly stable. The percentages of children who underwent surgery decreased from 17.3% in the first 6 years to 4.6% in the last 6 years (P < 0.001). Surgery was performed in 27.7% of CD and 5.3% of UC cases. The median age at diagnosis was 12.2 years for UC, 13.0 years for CD, 11.2 for IC, and 11.2 for PCD. At diagnosis, 48 children (7.5%) were 5 years of age or less, whereas most of the patients were 11 years of age or more (398 children, 62.3%). CONCLUSIONS: In Sweden, the incidence of UC has increased, whereas that of CD remains the same. A significant number of children were classified with IC and PCD. In most children, IBD was diagnosed when they were 11 years old or more, but some cases were detected even in those below 6 years of age. A decrease in the frequency of surgery occurred during the study.

Miniaturized direct on air sampling filter quantification of pollen allergens.

A recently in this journal reported luminescence immunoassay for the direct quantification of birch and grass pollen allergens on air sampling filters. DOSIS, has been miniaturized. By means of a commercially available chlorinated analogue of the previously used 1,2 dioxetane phosphate derivative as enzyme substrate, the air sampling filter diameter could be reduced from 25 mm to 13 mm. The procedure leads to a more than twenty times reduction of the previously reported limit of quantification for the grass pollen allergen.

Luminescence immunoassay of pollen allergens on air sampling polytetrafluoroethylene filters.

A new method for quantification of airborne birch and grass pollen allergens collected on porous polytetrafluoroethylene filters has been developed. In this method, the allergens firmly adsorbed to the sampling filter of 25 mm in diameter are reacted with specific antibodies conjugated to alkaline phosphatase, generating a matrix-bound allergen-antibody-phosphatase complex. The filter is then floated on a chemiluminescent enzyme substrate solution. The light intensity of the product is linearly related to the amount of allergen over a large mass range, 0-1000 SQ (1 SQ is about 250 pg of protein). This direct on sampling filter in solution (DOSIS) technique demonstrated intra-assay precisions between 6-16% and 11-15% for the levels of 1-100 SQ units of grass allergen Phl p 5 and 4-400 SQ units of birch allergen Bet v 1, respectively. The limits of quantification for the corresponding allergens were estimated to 0.5 and 2 SQ units. Application of DOSIS to analysis of the grass pollen allergen concentrations of outdoor air for 12 days in July 1998 revealed a correlation coefficient of 0.69 between pollen grain and allergen concentrations for the dry weather period. After rainy days large amounts of grass allergens were present even in the absence of pollen grains.

Quantification of birch and grass pollen allergens in indoor air.

Birch and grass pollen grains as well as pollen-derived small particles appear as potent allergens in the outdoor air during spring and summer. The occurrence of pollen allergens in indoor air, however, has not been studied in depth due to lack of suitable sampling and analytical methods. Herein, a recently reported "direct on sampling filter estimation" (DOSAFE) technique (Acevedo et al., 1998) has been validated for quantification of pollen allergens in indoor air using two school rooms and two office rooms as experimental models. Using DOSAFE and polyclonal antibodies against water extracts of pollen from Betula pendula and Phleum pratense L, we found that indoor air of school and office rooms carried substantial amounts of pollen allergens, expressed as SQ units, predominantly occurring as particles with smaller diameters than the pollen grains. In one school room the indoor air birch pollen allergen concentrations increased from 242 to 403 SQ units/m3 over the sampling period although the corresponding outdoor air concentrations decreased from 350 to 90 SQ units/m3. Electrostatic air cleaning in one office room reduced its grass pollen allergen concentrations by more than 95% to 0.02-0.34 SQ units/m3 as compared to the control room.

Methylomonas scandinavica sp. nov., a new methanotrophic psychrotrophic bacterium isolated from deep igneous rock ground water of Sweden.

Methane-utilizing bacteria were enriched from deep igneous rock environments and affiliated by amplification of functional and phylogenetic gene probes. Type I methanotrophs belonging to the genera Methylomonas and Methylobacter dominated in enrichment cultures from depths below 400 m. A pure culture of an obligate methanotroph (strain SR5) was isolated and characterized. Pink-pigmented motile rods of the new isolate contained intracytoplasmic membranes as stacks of vesicles, assimilated methane via the ribulose monophosphate pathway and had an incomplete tricarboxylic acid cycle. Phosphatidyl glycerol, methylene ubiquinone and cytochrome c552 were prevailing. The DNA G+C content is 53.3 mol %. Strain SR5 grew at temperatures between 5 and 30 degrees C with optimum at 15 degrees C, close to its in situ temperature. Analyses of 16S rRNA gene, whole cell protein, enzymatic and physiological analyses of strain SR-5 revealed significant differences compared to the other representatives of Type I methanotrophs. Based on pheno- and genotypic characteristics we propose to refer the strain SR5 as to a new species, Methylomonas scandinavica.

A zone immunoelectrophoresis assay method for quantification of apolipoprotein D in human cerebrospinal fluid.

A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in human unconcentrated cerebrospinal fluid (CSF). The apo D concentrations of samples of the serum, plasma and CSF were directly proportional to the migration distances of the corresponding zones of immunoprecipitates developed during electrophoresis in glass capillaries filled with antibody-containing agarose gel. A linear standard curve, between about 1 and 12 mg of apo D/1 was obtained using a commercial serum preparation. Seronorm, as apo D standard. The coefficients of variation of the ZIA were below 8% (n = 5 x 6) and 10% (n = 8) for within-run and between-run reproducibility, respectively. Quantification experiments with disulfide-reducing agent, mixtures of CSF and urine as well as frozen and stored CSF samples indicated parallelism between the precipitate-forming immunologic reactions of apo D in different sample matrices when performed with ZIA. Application of this method to quantify apo D of CSF and plasma samples from 51 normal healthy men aged 16-72 years yielded means +/- SD of 5.3 +/- 1.5 mg/l and 128.4 +/- 22.7 mg/l, respectively. No correlation was found between the CSF and plasma apo D concentrations.

Global analysis of the carbon starvation response of a marine Vibrio species with disruptions in genes homologous to relA and spoT.

The stringent control response, which involves a rapid accumulation of ppGpp, is triggered if the marine Vibrio sp. strain S14 is subjected to carbon and energy starvation. By means of high-resolution two-dimensional gel electrophoresis analysis, we addressed the role of the major ppGpp-synthesizing enzyme (RelA) in the regulation of the carbon starvation response of Vibrio sp. strain S14. The finding that a large number of the carbon starvation-induced proteins were underexpressed in the Vibrio sp. S14 relA mutant strain after the onset of glucose starvation suggests that a rapid accumulation of ppGpp is required for induction of many of the carbon starvation-induced proteins. However, it was also found that a majority of the carbon starvation-induced proteins were significantly less induced if the stringent control response was provoked by amino acid starvation. We therefore also addressed the notion that a carbon starvation-specific signal transduction pathway, complementary to the stringent control, may exist in Vibrio sp. strain S14. It was found that a majority of the proteins that were underexpressed in the relA mutant strain were also underexpressed in the Vibrio sp. S14 spoT mutant strain (csrS1). Interestingly, a large proportion of these underexpressed proteins were found to belong to a group of proteins that are not, or significantly less, induced by starvation conditions that do not promote starvation survival. On the basis of these observations and the finding that the csrS1 strain survives poorly but accumulates ppGpp in a fashion similar to the wild type during carbon and energy source starvation, the gene product of the csrS gene is suggested to be responsible for the mediation of a signal which is complementary to ppGpp and essential for the successful development of the starvation- and stress-resistant cell. This conclusion was also supported by experiments in which changes in phenotypic characteristics known to be induced during carbon starvation were studied. The starvation induction of the high-affinity glucose uptake system was found to be dependent on the csrS gene but not relA, and the synthesis of carbon starvation-specific periplasmic space proteins was dependent, at different times of starvation, on both the relA and the csrS gene products.

Purification and identification of allergenic alpha (2u)-globulin species of rat urine.

Amino-acid compositional and sequence analyses as well as mass spectrometric determinations of purified rat urine proteins, previously termed prealbumin and alpha(2)-euglobulin, have revealed a high homology between the two forms which have now been identified as alpha(2)-globulin species. The "prealbumin' fraction was found to correspond to alpha(2u)-globulin originating from salivary gland and the 'alpha(2)-euglobulin' fraction was identical with the major urinary protein (MUP) or alpha(2u)-globulin. The results indicate that the two major protein fractions of rat urine constitute different forms of the same parent protein, alpha(2u)-globulin, having no amino-acid sequence resemblance to prealbumin (transthyretin) of rat serum.

Incidence and prevalence of inflammatory bowel disease in children in south-western Sweden.

The incidence and prevalence of inflammatory bowel disease were estimated in all children less than 16 years of age living in the city of Göteborg and in three countries in South-Western Sweden, from 1983 to 1987. One hundred and thirty-two patients were classified according to set criteria into one of four diagnostic categories: ulcerative colitis, Crohn's disease, probable Crohn's disease and indeterminate colitis. The crude incidence of inflammatory bowel disease was 5.3 per 100,000 children per year and the prevalence 21.5 per 100,000 children. This study lends support to the hypothesis that Crohn's disease has increased among Swedish children. Crohn's disease now appears to be at least as common as ulcerative colitis. Thirty-five of 55 patients first classified as indeterminate colitis or probable Crohn's disease later fulfilled the criteria of ulcerative colitis or Crohn's disease during a mean follow-up period of 4.6 years. This study emphasizes the importance, in epidemiological studies of inflammatory bowel disease, of including those cases where a definite diagnosis of ulcerative colitis or Crohn's disease cannot be established initially and of re-evaluating the initial diagnosis regularly.

The DnaK homologue of the marine Vibrio sp. strain S14 binds to the unprocessed form of a carbon starvation-specific periplasmic protein.

The Escherichia coli DnaK homologue in Vibrio sp. strain S14 was shown to possess chaperone function for translocation during carbon starvation. This was demonstrated by using the method of co-immunoprecipitation. DnaK co-precipitated with the carbon starvation-specific periplasmic space protein Csp5 three hours after the onset of carbon starvation. Pulse-chasing of the protein with radiolabelled methionine followed by the addition of an excess of unlabelled methionine demonstrated that the Csp5 protein was translocated across the inner membrane. Only the cytoplasmic unprocessed precursor form of Csp5 co-precipitated with DnaK. The non-covalent binding between the two proteins was found to be ATP-dependent, as the addition of ATP released the interaction between DnaK and the precursor form of Csp5, as was shown on silver-stained SDS-polyacrylamide gels and by Western blot analysis. We suggest that DnaK maintains the carbon starvation-inducible protein Csp5 in a translocation-competent form in the cytoplasm.

Polymerization and fractionation of house dust mite allergen.

Glutaraldehyde polymerization and appropriate fractionation of allergens result in significantly decreased allergenicity with retained immunogenicity. In a manner similar to that employed with other allergens, we have prepared monomer dust mite (MDM) and polymerized dust mite (PDM). As with other polymerized allergens such as polymerized ragweed, PDM is more than 2 logs less allergenic than MDM as determined by cutaneous end point titration in sensitive patients. Moreover, PDM and MDM are similarly immunogenic in rabbits. As determined by inhibition studies (with rabbit antisera) PDM is antigenically complete and contains no new epitopes against which antibody was produced. In summary, we have prepared PDM which has the desired characteristics of reduced allergenicity, retained immunogenicity, antigenic completeness, and absence of new antigenic determinants.

How do non-differentiating bacteria adapt to starvation?

Non-differentiating bacteria adapt to starvation induced growth arrest by a complex turn-on/turn-off pattern of protein synthesis. This response shows distinct similarities with those of spore formation in differentiating organisms. A substantial amount of information on the non-growth biology of non-differentiating bacteria can be derived from studies on Vibrio strains. One important result is that carbon rather than nitrogen or phosphorus starvation leads to the development of a starvation and stress resistant cell in these organisms. Hence, we have attempted to characterize the carbon starvation stimulon. By the use of two-dimensional gel electrophoresis of pulse-labelled cells and transposon mutagenesis, using reporter gene constructs, the identity and function of some members of the carbon starvation stimulon have been elucidated. Moreover, regulatory genes of the starvation response have been identified with these techniques. Current studies primarily address the identity and function of these genes. The role of transcript modification and stability for both long term persistence during starvation as well as the efficient recovery of cells which occurs upon nutrient addition is also addressed. It is suggested that an understanding of the functionality of the translational machinery is essential for the understanding of these adaptive pathways. This contribution also discusses the diversity of the differentiation-like response to starvation in different bacteria and whether a general starvation induced programme exists.

Apolipoprotein D and alpha 1-microglobulin in human urine: effect of cadmium exposure.

Apolipoprotein D is a previously unrecognized urinary protein of unknown function which we have tested as a potential marker for kidney malfunction. This protein and alpha 1-microglobulin have been quantified by zone immunoelectrophoresis assay in urine samples from a group of eight men occupationally exposed to cadmium-containing welding fumes for many years. All these workers had highly elevated concentrations of urinary cadmium and indications of tubular proteinuria, as compared to a group of 50 apparently healthy normal men analyzed in parallel. The cadmium-exposed workers demonstrated three- and 15-fold average increases in apolipoprotein D and alpha 1-microglobulin, respectively, over normal values in urine, estimated both as excretion rates and as milligrams of protein per mmol of creatinine. All these increments were highly significantly different (P < 0.001) from the corresponding values of the reference group. Essentially the same results were obtained for each of the proteins from two independent consecutive samplings of the workers' urine. There were good linear (R = 0.70, 0.80) and logarithmic (R = 0.84, 0.81) correlations between the urinary concentrations of alpha 1-microglobulin and apolipoprotein D for both the reference and the study group. Although not as sensitive an indicator for tubular proteinuria as alpha 1-microglobulin, apolipoprotein D, being a storage-stable urinary protein, seems a valuable complement for the diagnosis of tubular malfunction.

Microheterogeneity of apolipoprotein D as revealed by electroblotting following isoelectric focusing in Immobiline DryPlates.

The microheterogeneity of apolipoprotein D was examined by a procedure involving, in sequences: (i) electrophoresis in an immobilized pH 4-7 gradient in an Immobiline DryPlate-polyacrylamide gel supplemented with Ampholine pH 5-7, (ii) covering of the gel with sodium dodecyl sulfate-containing agarose, (iii) electroblotting onto a polyvinylidene difluoride membrane and (iv) immunological identification. Seven isoforms were obtained with partially purified apolipoprotein D. Using this technique the apparent pI values at 15 degrees C for the isoforms were 4.57, 4.67, 4.78, 4.83 and 5.95, 6.06 and 6.19 (SD +/- 0.05 for all). Direct staining of the Immobiline DryPlate could not reveal the isoforms of partially purified apolipoprotein D.

Quantification of apolipoprotein D in human urine by zone immunoelectrophoresis assay: a methodological and clinical study.

A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in unconcentrated native human urine. A standard curve, linear between 1 and 8 mg apo D/l was obtained with ZIA. The relative coefficients of variation for this method were 5-9% (n = 15 x 6) with a mean +/- SD of 7 +/- 1.4% and below 11% (n = 6 x 15) for within-run and between-run reproducibility, respectively. Equal amounts of apo D in unconcentrated and diluted urines, in serum and of the purified protein produced the same zone migration distances indicating parallelism between the immunologic reactions of apo D in different sample matrixes. Storage experiments with normal urines demonstrated good stability of apo D in both acidic and alkalinized urine over at least 2 days at +5 degrees C and during several days at -20 degrees C to -40 degrees C. Using ZIA, urine samples from 50 normal healthy men aged 23-65 years were analyzed for apo D. Mean and SD were: 2.8 +/- 2.1 mg/l, 2.6 +/- 1.8 micrograms/min and 0.24 +/- 0.13 mg/mmol for concentration, rate of excretion and mass/creatinine concentration, respectively.