Ultrasonographic assessment of tendon thickness, Doppler activity and bony spurs of the elbow in patients with lateral epicondylitis and healthy subjects: a reliability and agreement study.

PURPOSE: Tennis elbow, also known as lateral epicondylitis (LE), is a common disorder often assessed by ultrasound. The aim of this study was to evaluate the ultrasonographic outcomes and methods used in LE research and clinical practice. MATERIALS AND METHODS: This study was designed as an intra- and interobserver reliability and agreement study. Ultrasonographic examination of the common extensor tendon of the elbow was performed. The intraobserver study examined tendon thickness twice in 20 right elbows from 20 healthy individuals at an interval of 7 to 12 days. The interobserver study examined tendon thickness, color Doppler activity, and bony spurs in 18 right elbows in 9 healthy individuals and 9 patients with LE. Two trained rheumatologists performed the interobserver examinations with the same scanner on the same day. The main outcomes were intra- and interclass correlation (ICC) and agreement. RESULTS: In the intraobserver study, the ICC with regard to tendon thickness ranged from 0.76 to 0.81, depending on the measurement techniques used. The agreement ranged from 0.06 to 0.13 mm. In the interobserver study, the tendon thickness ICC ranged from 0.45 to 0.65 and the agreement ranged from -0.17 to 0.13 mm. The ICC for color Doppler activity was 0.93, with agreement in 14/18 (78 %) of the cases. A perfect reliability was demonstrated for bony spurs, with an ICC of 1 and exact agreement in 18/18 (100 %) of the cases. CONCLUSION: Good to excellent reliability was obtained for all measurements. The ultrasonographic techniques evaluated in this trial can be recommended for use in both research and clinical practice.

Is the quality of drinking water a risk factor for self-reported forearm fractures? Cohort of Norway.

SUMMARY: Compared to pH ≥7.0 in Norwegian municipal drinking water, pH <7.0 increased the risk of forearm fractures in the population-based Cohort of Norway (CONOR; n = 127,272). The association was attenuated (p > 0.05) after adjustments for indicators of bacteria and organic matter, which may signify an association between poor drinking water and bone health. INTRODUCTION: The Norwegian population has the highest rate of fractures ever reported. A large variation in fracture rate both between and within countries indicates that an environmental factor, such as the quality of drinking water, could be one of the causes of the disparities. Our aim was to investigate a possible association between pH (an important parameter for water quality) and self-reported forearm fracture and to examine whether other water quality factors could account for this association. METHODS: Using Geographic Information Systems, information on the quality of drinking water was linked to CONOR (n = 127,272; mean age, 50.2 ± 15.8 years), a database comprising ten regional epidemiological health surveys from across the country in the time period 1994-2003. RESULTS: The highest risk of forearm fracture was found at a pH of around 6.75, with a decreasing risk toward both higher and lower pH values. The increased adjusted odds of forearm fracture in men consuming municipal drinking water with pH <7.0 compared to water with pH ≥7.0 was odds ratio (OR) = 1.19 (95 % CI, 1.14, 1.25), and the corresponding increased odds in women was OR = 1.14 (95 % CI, 1.08, 1.19). This association was attenuated (p > 0.05) after further adjustments for other water quality factors (color grade, intestinal enterococci, and Clostridium perfringens). CONCLUSIONS: Our findings indicate a higher risk of fracture when consuming water of an acidic pH; however, the risk does not only seem to be due to the acidity level per se, but also to other aspects of water quality associated with pH.

Outbreak of tularaemia in central Norway, January to March 2011.

From January to March 2011, 39 cases of tularaemia were diagnosed in three counties in central Norway: 21 cases of oropharyngeal type, 10 cases of glandular/ulceroglandular type, two of respiratory and two of typhoid type. Three cases were asymptomatic and clinical information was unavailable for one case. The mean age was 40.3 years (range 2-89 years). Thirty-four reported use of drinking water from private wells. An increased rodent (lemming) population and snow melting may have led to contamination of the wells with infected rodents or rodent excreta.

In vivo clearance and metabolism of recombinant activated factor VII (rFVIIa) and its complexes with plasma protease inhibitors in the liver.

INTRODUCTION: Recombinant activated factor VII (rFVIIa, NovoSeven®) is injected intravenously for the treatment of haemophilia patients with inhibitory antibodies. In plasma, rFVIIa forms complexes with protease inhibitors, primarily antithrombin III (ATIII). The liver is believed to be involved in clearance of rFVIIa, however, it is not known whether the liver is also involved for the clearance of the rFVIIa-ATIII complex. In this study, we explored the fate of intravenously injected rFVIIa from plasma to the hepatic lysosomes. MATERIALS AND METHODS: A novel method using magnetic chromatography was used to isolate catabolic organelle (CO) fractions from mouse liver following injection of superparamagnetic dextran (SPD)-coated iron oxide particles and rFVIIa. The effect of co-circulating SPD particles on rFVIIa pharmacokinetic (PK) parameters was evaluated by ELISA. Cryo-immuno transmission electron microscopy (TEM) was used to study hepatic distribution of SPD particles and rFVIIa. The isolated hepatic CO fractions were characterized using Western Blotting (WB). RESULTS: Cryo-immuno TEM of the liver confirmed hepatic co-localisation of SPD particles and rFVIIa in identical endosomes and lysosomes of both hepatocytes and Kupffer cells. SPD particles did not affect the PK parameters of rFVIIa. WB analysis of plasma and CO fractions detected rFVIIa as the full-length protein and also in high molecular weight (HMW) complexes with ATIII and α-2 macroglobulin (α-2M). CONCLUSIONS: Following injection, both hepatocytes and Kupffer cells appeared to be involved in the hepatic clearance and metabolism of both full-length rFVIIa and rFVIIa in complex with at least two plasma protease inhibitors; ATIII and α-2M.

Purification and characterization of a new recombinant factor VIII (N8).

SUMMARY: A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B-domain and a light chain of 79 kDa. The two chains are held together by non-covalent interactions. The four-step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800-9800 IU mg(-1). Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N-terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B-domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N-linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N-linked glycans are sialylated bi-antennary structures. An O-glycosylation site is present in the B-domain linker region. This site was glycosylated with a doubly sialylated GalNAc-Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well-characterized FVIII product with biochemical properties that equal other FVIII products.

Glycosylation analysis of gel-separated proteins.

Beyond the identification of proteins involved in a particular physiological situation, many aspects of proteomics require more detailed characterization of the proteins involved. Post-translational modifications (PTMs) of proteins are a common means to target proteins, regulate their activities and to mediate communication between proteins and cells. Owing to the much higher analytical complexity of glycan analysis compared to e.g. protein identification, PTM analysis in general and glycosylation analysis in particular is largely neglected in proteomics. In this review, the current technological status of global and site-specific glycosylation analysis of gel-separated proteins is described and the way in which the available technology can be employed in proteomics is critically discussed.

Identification of precursor forms of free prostate-specific antigen in serum of prostate cancer patients by immunosorption and mass spectrometry.

The structural features of the free prostate-specific antigen (F-PSA) present in human blood have not been clarified up to now, and it is, therefore, not known why F-PSA is not complexed by the protease inhibitors that are present in human blood in large amounts. This lack of information is mainly attributable to the low amount of F-PSA in serum, which makes the isolation and structural characterization very difficult, especially when only limited amounts of individual sera are available. It has now been demonstrated that F-PSA occurs as a mixture of different pro-PSA forms (zymogen forms) in the sera of prostate cancer patients, and that, in some of these sera, a form with the regular NH2 terminus of PSA is present as well. Among the five serum samples investigated, all contained the (-7), (-5), and (-4) pro-PSA forms, whereas the (-1) and (-2) forms were only present in three of them. These three samples also contained the form with the regular NH2 terminus. The (-3) and (-6) pro-PSA forms have not been detected thus far. The F-PSA has been isolated by immunosorption from the individual sera using streptavidin-coated magnetic beads. The pro-PSA forms were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry after producing peptides by endoproteinase from Lysobacter enzymogenes digestion of the SDS-PAGE-separated F-PSA band. The structural identity of the (-7)pro-PSA form was further proven by sequencing of that particular peptide using electrospray ionization quadrupole time-of-flight mass spectrometry.

RNA fragmentation studied in a matrix-assisted laser desorption/ionisation tandem quadrupole/orthogonal time-of-flight mass spectrometer.

We have studied the fragmentation behaviour of short, singly protonated oligoribonucleotides on a MALDI Qq-TOF instrument with the aim of using this instrumental set-up to characterise modifications of RNA molecules. Individual ion species from enzymatically generated mixtures were isolated in one quadrupole and subjected to collision-induced dissociation in a second quadrupole followed by separation of the resulting product ions in an orthogonal time-of-flight mass analyser. Complex spectra were generally observed with nearly all types of cleavages along the phosphodiester backbone and of the N-glycosidic bonds (and combinations of these) occurring, albeit at different relative intensities. The most labile part of the backbone was found to be the 5'-P-O bond, resulting in c- and y-ions. Loss of neutral cytosine and guanine occurred equally often, whereas neutral loss of adenosine was less prevalent. Loss of uracil, either neutral or charged species, was not observed. Because the fragmentation pattern observed here is significantly different from what has been reported for singly protonated oligodeoxyribonucleotides, we suggest that the 2'-substituent in the sugar plays a central role in the fragmentation mechanisms of nucleic acids. Finally, we used the acquired knowledge about oligoribonucleotide fragmentation to characterise an in vivo methylated oligoribonucleotide by tandem mass spectrometry.

Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis.

In proteomics it is essential to be able to detect proteins separated by gel electrophoresis at high sensitivity. Silver staining is currently the most popular method. Here we present silver staining protocols that are optimized for staining sensitivity, peptide recovery and compatibility with digestion and mass spectrometry.

Protein analysis using enzymes immobilized to paramagnetic beads.

A new method combining protein chemistry with enzymes immobilized to paramagnetic beads is presented. The immobilized enzymes can substitute for regular enzymes in a number of protein chemistry protocols, resulting in faster reaction times, less sample contamination, and improved interfacing to modern procedures, like mass spectrometry. Trypsin was used as a model enzyme to test the amount of protein coupled to glass beads and the degree of autodigestion when analyzed by MALDI-MS and HPLC. Immobilization of trypsin resulted in digestions comparable with standard solution digestions using fetuin as a model substrate. Furthermore, fetuin was used to test the stability of the enzyme-coated beads. No apparent loss of enzyme activity was observed after 10 times reuse of trypsin-coated beads. Immobilization of exo- and endoglycosidases to paramagnetic beads resulted in high sensitivity, faster sequential glycosidase digestion of glycopeptides, and reduced sample contamination. All digestions could be performed in less than 24 h, when a tryptic glycopeptide from human lung proteinosis surfactant protein A was used as model compound.

Water chlorination and birth defects.

Chlorination of drinking water that contains organic compounds leads to the formation of by-products, some of which have been shown to have mutagenic or carcinogenic effects. As yet, too little is known about the possible teratogenic effects on the human fetus. We linked the Norwegian waterwork registry, containing 1994 data on chlorination practice and color (an indicator for natural organic matter), with the Medical Birth Registry for 1993-1995. The proportion of the population exposed to chlorination and a weighted mean color number in drinking water was computed for each municipality. Among 141,077 births, 2,608 (1.8%) had birth defects. In a comparison between exposed (high color; chlorination) and reference groups (low color; no chlorination), the adjusted odds ratio was 1.14 (0.99-1.31) for any malformation, 1.26 (0.61-2.62) for neural tube defects, and 1.99 (1.10-3.57) for urinary tract defects. This study provides further evidence of the role of chlorination of humic water as a potential cause of birth defects, in a country with relatively low levels of chlorination byproducts.

Thermal instability of the trimeric structure of the N-terminal propeptide of human procollagen type I in relation to assay technology.

The N-terminal propeptide of procollagen type I (PINP) appeared in two peaks after size chromatography. The high-molecular weight form was transformed to the low-molecular weight form during incubation at 37 degreesC, whereas the low-molecular weight form remained unchanged. The PINP concentrations in amniotic fluid and sera remained unchanged during 37 degreesC incubation when measured using an ELISA; however, concentrations decreased by 89-93% when measured using an RIA. The ELISA:RIA ratio varied from 1.1 to 2.9 in these fluids because of different size distributions and the inability of the RIA to measure the low-molecular weight form. Thermal transition of the high-molecular weight form caused a change in its elution volume but did not change its migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectrometry revealed identical results for both forms. We reached the following conclusions: (a) the trimeric structure of PINP is unstable at 37 degreesC; (b) the two molecular forms represent intact alpha1 chains in trimeric and monomeric forms; (c) thermal transition is an ongoing in vivo process; and (d) this is important in the choice of assay technology.

Characterization of intramolecular disulfide bonds and secondary modifications of the glycoprotein from viral hemorrhagic septicemia virus, a fish rhabdovirus.

Viral hemorrhagic septicemia virus (VHSV) infections cause high losses in cultured rainbow trout in Europe. Attempts to produce a recombinant vaccine based on the transmembrane glycoprotein (G protein) have indicated that proper folding is important for the antigenicity and immunogenicity of the protein. The present study was initiated to identify the disulfide bonds and other structural aspects relevant to vaccine design. The N-terminal amino acid residue was identified as being a pyroglutamic acid, corresponding to Gln21 of the primary transcript. Peptides from endoproteinase-degraded G protein were analyzed by mass spectrometry before and after chemical reduction, and six disulfide bonds were identified: Cys29-Cys339, Cys44-Cys295, Cys90-Cys132, Cys172-Cys177, Cys195-Cys265, and Cys231-Cys236. Mass spectrometric analysis in combination with glycosidases allowed characterization of the glycan structure of the G protein. Three of four predicted N-linked oligosaccharides were found to be predominantly biantennary complex-type structures. Furthermore, an O-linked glycan near the N terminus was identified. Alignment of the VHSV G protein with five other rhabdovirus G proteins indicates that eight cysteine residues are situated at conserved positions. This finding suggests that there might be some common disulfide bonding pattern among the six rhabdoviruses.

Glycosylation analysis and protein structure determination of murine fetal antigen 1 (mFA1)--the circulating gene product of the delta-like protein (dlk), preadipocyte factor 1 (Pref-1) and stromal-cell-derived protein 1 (SCP-1) cDNAs.

By means of sequence analysis, murine fetal antigen 1 (mFA1) isolated from Mus musculus amniotic fluid was shown to be the circulating protein of the delta-like protein, stromal-cell-derived protein 1 (SCP-1) and preadipocyte factor 1 (Pref-1) gene products. The protein contains 36 cysteine residues arranged in six epidermal-growth-factor-like domains. The purification of several C-terminal peptides of varying lengths showed mFA1 to be C-terminal heterogeneous. O-linked glycosylations of the NeuNAc alpha2-3Gal beta1-3(NeuNAc alpha2-6)GalNAc type were present on all C-terminal peptides at residues Thr235, Thr244 and Thr248, although glycosylation on Thr244 was only partial. Three N-linked glycosylations were localized in mFA1 (Asn77, Asn142 and Asn151), two of which (Asn142 and Asn151) were in the unusual Asn-Xaa-Cys motif. Fucosylated biantennary complex-type and small amounts (less than 5%) of triantennary complex-type structures were identified on the glycosylated asparagine residues using sequential exoglycosidase and endoglycosidase digestions combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The presence of O-linked monosaccharides (glucose attached to Ser71, Ser193 and fucose at Thr201) was tentatively ascertained by combining Edman degradation and MALDI-MS. The results presented shows mFA1 to be the circulating heterogeneous cleavage products of the membrane-bound protein encoded by the murine cDNAs dlk, pref-1 and SCP-1.

Fetal antigen 1 (FA1), a circulating member of the epidermal growth factor (EGF) superfamily: ELISA development, physiology and metabolism in relation to renal function.

We describe an ELISA technique for quantification of fetal antigen 1 (FA1), a glycoprotein belonging to the EGF-superfamily. The ELISA is based on immunospecifically purified polyclonal antibodies and has a dynamic range of 0.7-5.3 ng/ml, intra- and inter-assay C.V.s of less than 3.2% and an average recovery of 105% in serum and 98% in urine. Comparison of FA1 in amniotic fluid, serum and urine revealed parallel titration curves, identical elution volumes following size chromatography, immunological identity and similar profiles when analysed by MALDI-MS. The reference interval for serum FA1 was 12.3-46.6 ng/ml and the levels were 10 times higher in patients with renal failure. FA1 showed no diurnal variation, no variation during the menstrual cycle and was not influenced by the acute phase reaction. In humans (n = 10) the renal clearance of FA1 was 11 ml/min and an identical high renal clearance was found in rats when expressed per 100 g body weight. In rats the initial increase in serum FA1 was 10 ng/ml/h following bilateral nephrectomy, explaining the increased serum concentrations of FA1 observed in patients with renal failure.