A variant near the interleukin-6 gene is associated with fat mass in Caucasian men.

CONTEXT: Regulation of fat mass appears to be associated with immune functions. Studies of knockout mice show that endogenous interleukin (IL)-6 can suppress mature-onset obesity. OBJECTIVE: To systematically investigate associations of single nucleotide polymorphisms (SNPs) near the IL-6 (IL6) and IL-6 receptor (IL6R) genes with body fat mass, in support for our hypothesis that variants of these genes can be associated with obesity. DESIGN AND STUDY SUBJECTS: The Gothenburg Osteoporosis and Obesity Determinants (GOOD) study is a population-based cross-sectional study of 18- to 20-year-old men (n=1049), from the Gothenburg area (Sweden). Major findings were confirmed in two additional cohorts consisting of elderly men from the Osteoporotic Fractures in Men (MrOS) Sweden (n=2851) and MrOS US (n=5611) multicenter population-based studies. MAIN OUTCOME: The genotype distributions and their association with fat mass in different compartments, measured with dual-energy X-ray absorptiometry. RESULTS: Out of 18 evaluated tag SNPs near the IL6 and IL6R genes, a recently identified SNP rs10242595 G/A (minor allele frequency=29%) 3' of the IL6 gene was negatively associated with the primary outcome total body fat mass (effect size -0.11 standard deviation (s.d.) units per A allele, P=0.02). This negative association with fat mass was also confirmed in the combined MrOS Sweden and MrOS US cohorts (effect size -0.05 s.d. units per A allele, P=0.002). When all three cohorts were combined (n=8927, Caucasian subjects), rs10242595(*)A showed a negative association with total body fat mass (effect size -0.05 s.d. units per A allele, P<0.0002). Furthermore, the rs10242595(*)A was associated with low body mass index (effect size -0.03, P<0.001) and smaller regional fat masses. None of the other SNPs investigated in the GOOD study were reproducibly associated with body fat. CONCLUSIONS: The IL6 gene polymorphism rs10242595(*)A is associated with decreased fat mass in three combined cohorts of 8927 Caucasian men.

Autoantibodies associated with congenital heart block.

Congenital heart block is the most severe manifestation of neonatal lupus syndrome. It is a passively acquired disease where transplacental passage of maternal autoantibodies is associated with irreversible damage of the foetal cardiac conduction system. It is well established that the condition, in the absence of structural abnormalities, is strongly associated with maternal autoantibodies to the Ro/La antigens. More specifically the disease has been closely linked to antibodies to the Ro52 component of the antigen complex. Congenital heart block constitutes a unique model where specific autoantibodies target and mediate organ-specific disease. A wide panel of maternal antibodies has been discussed in literature in association with the disease and are described in this review.

Variants of the interleukin-1 receptor antagonist gene are associated with fat mass in men.

CONTEXT: Immune functions seem to have connections to variations in body fat mass. Studies of knockout mice indicate that endogenous interleukin (IL)-1 can suppress mature-onset obesity. OBJECTIVE: To systematically investigate our hypotheses that single-nucleotide polymorphisms (SNPs) and/or haplotypes variants in the IL-1 gene system are associated with fat mass. SUBJECTS: The Gothenburg osteoporosis and obesity determinants (GOOD) study is a population-based cross-sectional study of 18-20 year-old men (n=1068), from Gothenburg, Sweden. Major findings were confirmed in elderly men (n=3014) from the Swedish part of the osteoporotic fractures in men (MrOS) multicenter population-based study. MAIN OUTCOME MEASURE: The genotype distributions and their association with body fat mass in different compartments, measured with dual-energy X-ray absorptiometry (DXA). RESULTS: Out of 15 investigated SNPs in the IL-1 receptor antagonist (IL1RN) gene, a recently identified 3' untranslated region C>T (rs4252041, minor allele frequency=4%) SNP was associated with the primary outcome total fat mass (P=0.003) and regional fat masses, but not with lean body mass or serum IL-1 receptor 1 (IL1RN) levels. This SNP was also associated with body fat when correcting the earlier reported IL1RN+2018 T>C (rs419598) SNP (in linkage disequilibrium with a well-studied variable number tandem repeat of 86 bp). The association between rs4252041 SNP and body fat was confirmed in the older MrOS population (P=0.03). The rs4252041 SNP was part of three haplotypes consisting of five adjacent SNPs that were identified by a sliding window approach. These haplotypes had a highly significant global association with total body fat (P<0.001). None of the other investigated members of the IL-1 gene family displayed any SNPs that have not been described previously to be significantly associated with body fat. CONCLUSIONS: The IL1RN gene, shown to enhance obesity by suppressing IL-1 effects in experimental animals, have not [corrected] previously described gene polymorphisms and haplotypes that are associated with fat, but not lean mass in two populations of men.

Antibodies to amino acid 200-239 (p200) of Ro52 as serological markers for the risk of developing congenital heart block.

Maternal autoantibodies to the p200-epitope of Ro52 have been suggested to correlate with development of congenital heart block. The aim of the present study was to evaluate the clinical relevance and predictive value of p200-antibodies in high-risk pregnancies. Sera from 515 Finnish, Swedish and American women were included in the study. Sera originated from 202 mothers with an infant affected by second- or third-degree atrioventricular block (AVB), 177 mothers with rheumatic disease having infants with normal heart rate and female blood donors (n = 136). A novel serological assay for Ro52 p200-antibodies with intra- and inter-assay variability of 3% and 3.8% respectively was developed. Mothers of children affected by AVB II-III had significantly higher p200-antibody levels than mothers with rheumatic disease having children with normal heart rate (P < 0.001). In the Swedish cohort, a distinction between foetuses with normal conduction, AVB I, AVB II and III was possible. A significant difference in anti-p200 levels between AVB I and AVB II-III groups compared with foetuses with normal conduction (P < 0.05 and P < 0.01) was observed. Using p200-antibodies as a second step analysis in Ro52-positive pregnancies increased the positive predictive value for foetal cardiac involvement (AVB I, II or III) from 0.39 (0.27-0.51) to 0.53 (0.37-0.68). In conclusion, Ro52 p200-antibodies may occur in women with unaffected children, but levels are significantly higher in mothers of children with congenital heart block and are suggested as a relevant marker in evaluating the risk for foetal AV block.

Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries.

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

Modeling of high cell density fed batch cultivation.

Escherichia coli was grown in carbon- and energy source-limited fed batch cultures to study the effect of osmotic stress and different feed rates on the growth kinetics. An unstructured model based on the linear equation for substrate consumption provided an adequate description of the bacterial growth during the first phase of biomass production (20 h), except for cultures exposed to osmotic stress by the addition of 0.5 M NaCl. The addition of salt to the culture media had a large effect on the energetics, that could not simply be described in terms of an increased maintenance requirement. In the later phase of growth, an extensive decline in viability for all cultures was observed. Coincidentally, the specific sugar uptake rate approached a lower limit. It is concluded that the total obtainable biomass in a fed batch culture is strongly affected by the magnitudes of the substrate feed rate and the ionic strength of the culture medium.

The use of fed batch cultivation for achieving high cell densities in the production of a recombinant protein in Escherichia coli.

The production of the fusion protein staphylococcal protein A/E. coli beta-galactosidase in Escherichia coli was studied in batch and fed batch cultivations. Batch cultivation of a recombinant E. coli strain yielded a final cell dry weight of 16.4 g l-1 with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the cell dry weight. Fed batch cultivation made it possible to increase the final cell dry weight to 77.0 g l-1. The intracellular product concentration (25%) was lower as compared to batch cultivation resulting in a total concentration of recombinant protein of 19.2 g l-1.

Prognostic information from on-line vectorcardiography in acute myocardial infarction.

The present study assesses the prognostic information from continuous on-line vectorcardiography in patients with acute myocardial infarction (AMI). A series of 203 patients with AMI were studied. Vectorcardiographic (VCG) recordings were obtained continuously for 24 hours. Analysis was performed on-line with the commercial system MIDA CoroNet. QRS vector difference (QRS-VD), ST change vector magnitude (STC-VM), and ST vector magnitude (ST-VM) were monitored. Patients were followed for 538 +/- 220 days. During follow-up, 36 patients died from cardiac causes and 38 patients had reinfarction. A significantly higher occurrence of transient VCG changes (QRS-VD, STC-VM, and ST-VM; p < 0.001) was seen in patients who died from cardiac causes or experienced either cardiac death or reinfarction at follow-up. The end value for QRS-VD was higher in patients who died from cardiac causes and correlated with the maximal value for creatine kinase when all patients were considered (r = 0.66; p < 0.001). Significantly lower mortality was seen in patients with VCG trend curves suggestive of coronary reperfusion (p < 0.01). In multivariate analysis, occurrence of transient changes in STC-VM, high QRS-VD end value, and VCG trend curves not suggestive of reperfusion gave additional prognostic information beyond that of age, gender, maximal creatine kinase value, heart size on chest x-ray, occurrence of ventricular fibrillation during hospitalization, and the inability to perform exercise tests. VCG monitoring during the first 24 hours of hospitalization for an AMI is a promising method for early detection of patients with increased risk for subsequent cardiac death or reinfarction.

Expression and characterization of a tripartite fusion protein consisting of chimeric IgG-binding receptors and beta-galactosidase.

Using protein engineering, a tripartite fusion protein was constructed consisting of five IgG-binding regions of protein A from Staphylococcus aureus, two IgG-binding regions of protein G from Streptococcus strain G148 and beta-galactosidase from Escherichia coli. The resulting protein lacks the serum albumin binding regions of native protein G. The fusion protein, which is a tetramer of approximately 660 kDa, was designed as a tool for immunological assays taking advantage of its broad spectrum of antibody affinity. The gene was placed under control of two promoters, the PR promoter and the lac UV5 promoter and the expression from the two promoters was studied in a bioreactor. Induction of the PR promoter gave an intracellular product concentration corresponding to 20% of the cell dry weight. By utilizing the properties of beta-galactosidase, the protein was purified by extraction in an aqueous two-phase system. The fusion protein was not proteolytically degraded during the cultivation and purification steps. The biological activity of all three parts of the protein was demonstrated with a competitive ELISA.

Fluorescence studies on plasminogen activator inhibitor 1: reactive centre cysteine mutants remain active after fluorophore attachment.

To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes. After expression in E. coli and purification to homogeneity, both of the mutant proteins were found to have similar biochemical characteristics as wild type PAI-1 (wtPAI-1). Following labelling with 4-chloro-7-nitrobenzofurazan (NBD) and 2-(4'-iodoacetamido-anilino)naphtalene-6-sulfonic acid (IAANS) the mutant inhibitors showed similar inhibitory activities and heat stability as wtPAI-1. The purified complex between uPA and NBD-labelled P3cys mutant was found to be extremely stable, suggesting that no slow cleavage or reversible reaction occurs in complexes that have been properly formed. The rate of labelling of both mutants was decreased when the mutants were in the latent form indicating that these cysteine residues may be less accessible in the latent configuration. The PAI-1 mutants labelled with both NBD and IAANS could convert from the active to the latent form, but P3cys labelled with the larger IAANS chromophore showed a two fold decrease in the rate of conversion to latency, suggesting that a large chromophore in the P3 position may interfere with the active to latent conversion. The fluorescence spectra of the two NBD labelled mutants were similar, but the intensity was three times higher for the P3cys mutant than for P18cys. No significant spectral changes could be seen when the P3cys mutant was transferred to latency. In contrast, the P18cys mutant showed a major change in the excitation spectra characteristic of migration of the NBD chromophore from a thiol to an amine. Complex formation with uPA had no effect on the fluorescence spectrum of P18cys-NBD while the spectrum of P3cys-NBD revealed changes consistent with a restriction of the mobility of NBD probe in the uPA-PAI-1 complex.

Cell segregation and lysis have profound effects on the growth of Escherichia coli in high cell density fed batch cultures.

Cell segregation into nondividing states and lysis was found to dominate the growth behavior of high cell density fed batch cultures of Escherichia coli. When the specific growth rate declined below a critical value, the biomass production, oxygen consumption, and carbon dioxide formation rates declined sharply. Concomitantly, an extensive loss of colony-forming ability (cfu) and accumulation of extracellular proteins was observed. A segregated model that considered different physiological states, including dividing, nondividing, and lysed cells, was developed and applied to experimental data from high cell density cultures of E. coli.

Concentrations and biomagnification of polychlorinated naphthalenes in black cormorants Phalacrocorax carbo sinensis from the Gulf of Gdansk, Baltic Sea.

Black cormorants, Phalacrocorax carbo sinensis (the breast muscles and liver) originating from the colony near Katy Rybackie on the south coast of the Gulf of Gdansk, Baltic Sea, were collected in 1992 and analysed for polychlorinated naphthalenes. PCNs were determined employing a multi-residue procedure including a non-destructive wide-bore open-tube extraction step, cleanup using semipermeable polyethylene membrane, HPLC fractionation of planar compounds on activated carbon column and final separation and identification and quantification with HRGC/HRMS. Tetra- to hepta-CNs were found in all samples examined, and penta- and next tetra-CNs were dominating homologue groups. Dominating members in the fingerprint of PCNs in black cormorants were 1,3,5,7-T4CN (no. 42); 1,2,3,5,7-/1,2,4,6,7-P5CN (no. 52/60); 1,2,4,6,8-P5CN (no. 61); 1,2,3,4,6,7-/1,2,3,5,6,7-H6CN (no. 66/67); 1,2,4,6-/1,2,4,7-/1,2,5,7-T4CN (no. 33/34); 1,2,4,5,7-P5CN (no. 58); and 1,2,4,7,8-P5CN (no. 62). When related to potential food items, black cormorants biomagnify in their body many PCNs and the congeners no. 42 and 66/67 show highest biomagnification factor (BMF) values.

Concentrations and spatial variations of cyclodienes and other organochlorines in herring and perch from the Baltic Sea.

Herring (Clupea harengus) and perch (Perca fluviatilis) were collected in the northern and southern Baltic Sea and analyzed for the presence of the cyclodiene pesticides chlordane (CHL), heptachlor, aldrin, dieldrin, endrin, isodrin, endosulfan and mirex, as well as other organochlorine contaminants, hexachlorocyclohexanes (HCHs), DDTs, hexachlorobenzene (HCBz) and polychlorinated biphenyls (PCBs) in order to investigate concentrations, accumulation and differences in geographical distribution. In the northern part of the Baltic Sea, Gulf of Bothnia, herring were collected at two pelagic stations, one in the Bothnian Bay (BB) and the other in the Bothnian Sea (BS), respectively; perch were collected at four coastal locations along the Swedish coast. All these locations were selected to represent background areas except one in the vicinity of an industrialised and contaminated area. Both specimens were also caught in the southern part of the Baltic Sea, in the Gulf of Gdansk (GG), Poland, a potentially highly polluted area. From the eight cyclodiene pesticides analyzed, three were detected in herring and perch samples, including 12 different CHL-related compounds, dieldrin and mirex. To our knowledge, it is the first time that mirex has been detected in samples from the Baltic Sea. Neither heptachlor, aldrin, endrin, isodrin nor endosulfan were found. However, HCHs, DDTs, HCBz and PCBs were found in every sample investigated, and the concentrations ranged e.g. for the cyclodiene chemicals dieldrin and CHL-related compounds from 30 to 170 ng/g lipid and for PCBs from 360 to 5400 ng/g lipid, both fish species included. Differences in contamination burden between the sites can be seen, e.g. for herring the BB and GG locals were similar, and generally lower than BS for all chemicals except that of DDT where GG was the highest. For the perch samples the industrialised location had markedly higher concentrations of HCBz and PCBs than the other locations. This species also identifies GG as the most DDT contaminated site among the three studied areas.

Concentrations and biomagnification of 17 chlordane compounds and other organochlorines in harbour porpoise (Phocoena phocoena) and herring from the southern Baltic Sea.

Four species of harbour porpoise (Phocoena phocoena) and one herring (Clupea harengus) sample from the southern Baltic Sea were analysed in an attempt to study the concentration and biomagnification of 17 chlordane related compounds (CHLs) including 12 components present in technical chlordane, the toxic metabolites oxychlordane and cis-hepatchlorepoxide and the photoconversion products photoheptachlor and two photo-cis-chlordanes. The concentration and biomagnification ability of CHLs were also compared to other organochlorines such as HCHs (hexachlorocyclohexanes), hexachlorobenzene, DDTs, dieldrin, mirex and PCBs (polychlorinated biphenyls). Of the CHLs analysed, 16 were detected in porpoise and 15 in herring including the photoconversion products. In both species the highest concentrations were found for PCBs and DDTs. The concentration of PCBs and CHLs in porpoise varied from 5700-16,000 and 470-1250 ng/g lipid, and in herring from 1300 and 49 ng/g lipid, respectively. The biomagnification factor (BMF: concentration in organism/concentration in food; all lipid normalized) in porpoise was found to be high for CHLs followed by dieldrin and lowest for HCHs. Among the CHLs, a big variation of BMF (BMF range approximately 1-50) was found e.g., the nonachlorinated compounds biomagnified to the highest degree followed by cis-heptachlorepoxide, photoheptachlor and oxychlordane.

[The contamination of white-tailed sea eagles with organochlorines in Poland]. / Skazenie bielików zwiazkami chloroorganicznymi w Polsce.

The residues of PCBs, DDTs, HCHs, HCBz, PCBz, CHLs, aldrin, dieldrin, endrin, isodrin, endosulfan 1, endosulfan 2, mirex, TCPM-H and TCPM-OH were determined in breast muscles and an egg of white-tailed sea eagles collected in Poland in 1991-1995. The method of measurement was capillary gas chromatography and low resolution mass spectrometry (HRGC/LRMS) after a non-destructive extraction, clean-up and fractionation of the sample. Only aldrin, endrin, endosulfan 1 and endosulfan 2 were absent in birds and egg examined. Some of the adult white-tailed sea eagles collected dead from the coastal area of the Baltic Sea still remain relatively high contaminated with organochlorines, and the concentrations of PCBs and DDTs in those birds ranged between 2300-2600 and 490-2000 micrograms/g lipids, respectively. In dead egg concentration of PCBs was 390 micrograms/g lipids (25 micrograms/g wet weight), while of DDTs 270 micrograms/g lipids (18 micrograms/g w.w.).

[Compounds from the chlordane group in adipose tissue of harbour seals from the Gulf of Gdansk]. / Zwiazki z grupy chlordanu w tkance tluszczowej morswinów z Zatoki Gdanskiej.

The composition and the concentration of chlordanes were shown in blubber of harbour porpoises collected from the Gulf of Gdansk, Baltic Sea, in 1991-1992. Chlordanes were determined using capillary gas chromatography and low resolution mass spectrometry (HRGC/LRMS). The range of CHLs concentration in blubber was between 420-1000 ng/g wet weight (470-1300 ng/g lipid weight). The composition of CHLs residues was similar in males and females, with trans-nonachlor (37%) as a most abundant compound, and followed by heptachlor epoxide (15%), cis-chlordane (14%), cis-nonachlor (14%), oxychlordane (11%), MC6 (3%), MC5 (2%), trans-chlordane, MC7, U82 and MC4 (< 1%).

[Residues of chlordane in fish in the Gulf of Gdansk]. / Pozostalosci chlordanu w rybach w Zatoce Gdanskiej.

Concentrations, composition and spatial variations of the residues of the pesticide Chlordane were determined in several species of fish caught in Gulf of Gdansk. The residues of Chlordane (cis-i trans-chlordane, cis-i trans-nonachlor, oxychlordane, heptachlor, heptachlor epoxide, MC4, MC5, MC6, MC7, U82 and U83) were found in all fish examined, however, the concentrations noted were low, i.e. from 0.40 to 12 ng/g wet weight. Among the Chlordane constituents and metabolites determined trans-naonachlor, cis-chlordane, oxychlordan, heptachlor epoxide, cis-nonachlor, MC5, MC6 and trans-chlordane were dominated, and MC4, MC7, U82 and U83 were minor compounds. No heptachlor residues were found in fish examined. A small concentrations and specific composition of the residues of Chlordane and its metabolites determined in fish from the Gulf of Gdansk do indicate on a distant sources of pollution with that pesticide--mainly transported and deposited via the atmosphere.

[Dieldrin, aldrin, endrin, isodrin, endosulfan 1 and 2 on fish in the Gulf of Gdansk]. / Dieldryna, aldryna, endryna, izodryna, endosulfan 1 i 2 w rybach w Zatoce Gdanskiej.

The residues of dieldrin, aldrin, endrin, isodrin, endosulfan 1 and 2 has been determined in a several species of fish caught in the Gulf of Gdansk in 1992. The method of measurement was capillary gas chromatograph and low resolution mass spectrometry (HRGC/LRMS) after a nondestructive extraction and clean-up step with a further fractionation of the extract on Florisil column. Apart from dieldrin no other cyclodiene pesticides studied were found in fishes in detectable amounts, and for dieldrin concentrations ranged from 0.84 to 6.6 ng/g wet weight.

[DDT and it metabolites in fish in the Gulf of Gdansk]. / DDT i jego metabolity w rybach w Zatoce Gdanskiej.

The residues of DDT and its metabolites (DDTs; p,p'-DDT, o,p'-DDT, p,p'-DDD, o,p'-DDD, p,p'-DDE, o,p'-DDE i p,p'-DDMU) has been determined in ten species of edible fish caught in the Gulf of Gdansk in 1992. The method of measurement was capillary gas chromatography and low resolution mass spectrometry (HRGC/LRMS) after a nondestructive extraction and clean-up step with a further fractionation of the extract on Florisil column. All fish examined contained detectable residues of DDTs, and the concentrations ranged from 28 to 310 ng/g wet weight. o,p'-DDT accounted from 0.4 to 2.5% to DDTs content. The residue concentration of DDTs in herring (110 ng/g wet weight and 1100 ng/g lipid weight) in 1992 was threefold lower than in the years 1979-1983 and fourteen fold lower than in 1969-1973.