Feeding flaxseed oil but not secoisolariciresinol diglucoside results in higher bone mass in healthy rats and rats with kidney disease.

Flaxseed's oil and lignan, secoisolariciresinol diglucoside (SDG), are implicated in attainment of health and treatment of renal injury and osteoporosis. To test for these benefits, weanling Han:SPRD-cy rats (n=171) with or without kidney disease were randomized to diets made with either corn oil or flaxseed oil and with or without SDG for 12 weeks. In females, weight was lower with the SDG diet. In males fed flaxseed oil, lean mass was higher and fat % was lower. In both sexes, fat % was lower in diseased rats. Bone mineral content (BMC) and density were higher in rats fed flaxseed oil and lower in diseased rats, additionally; BMC was lower in SDG-supplemented females. The benefit of flaxseed oil on body composition is sex specific but the effect on bone mass is not. Lastly, reduced weight due to early rat kidney disease is not due to loss of lean body mass.

Free energy barrier estimation of unfolding the alpha-helical surfactant-associated polypeptide C.

Molecular dynamics simulations were conducted to estimate the free energy barrier of unfolding surfactant-associated polypeptide C (SP-C) from an alpha-helical conformation. Experimental studies indicate that while the helical fold of SP-C is thermodynamically stable in phospholipid micelles, it is metastable in a mixed organic solvent of CHCl3/CH3OH/0.1 M HCl at 32:64:5 (v/v/v), in which it undergoes an irreversible transformation to an insoluble aggregate that contains beta-sheet. On the basis of experimental observations, the free energy barrier was estimated to be approximately 100 kJ/mole by applying Eyring's transition state theory to the experimental rate of unfolding [Protein Sci 1998;7:2533-2540]. These studies prompted us to carry out simulations to investigate the unwinding process of two helical turns encompassing residues 25-32 in water and in methanol. The results give an upper bound estimation for the free energy barrier of unfolding of SP-C of approximately 20 kJ/mole. The results suggest a need to reconsider the applicability of a single-mode activated process theory to protein unfolding.

NMR studies of the sporulation protein SpoIIAA: implications for the regulation of the transcription factor sigmaF in Bacillus subtilis.

SpoIIAA participates in a four-component mechanism for phosphorylation-dependent transcription control at the outset of sporulation. We report the refinement of the solution structure of SpoIIAA by using the automated iterative NOE assignment method ARIA. To complement the structural data, the protein dynamics were determined by measuring the T1, T2 and NOE of the backbone 15N-nuclei. The refined structure permits a discussion of the structural features that are important for the function of SpoIIAA in the regulation of the sporulation sigma factor sigmaF, and for homologous regulatory pathways present in B. subtilis and in other bacilli.

Testing of a Chemiluminescence Enzyme Immunoassay for selective detection of E. Coli O157 from ground beef samples.

The aim of this study was to evaluate a Chemiluminescence Enzyme Immunoassay (CLIA) developed for the detection of E. coli O157:H7, using different E. coli O157 serotypes. The sensitivity and specificity of the kit were determined from the tenfold dilutions of the 24-hour broth cultures of the test strains. According to the results obtained in this trial, the sensitivity of the kit is 10(3)-10(4) cells ml-1, and it is specific for E. coli O157. Twenty-five g ground raw beef samples were prepared and inoculated with E. coli O157:H7 at different CFU g-1. The samples were incubated in 225 ml of modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 4 h and the immunoassays were performed following the instructions of the manufacturer. According to the results obtained by the CLIA test 10(1)-10(2) E. coli O157 g-1 can be detected from the sample. So this kit seems to be suitable for screening the samples before selective cultivation of E. coli O157:H7.

Some NMR experiments and a structure determination employing a [15N,2H] enriched protein.

We present the results of studies of an aqueous sample of a highly [15N,2H] enriched protein, the SH3 domain from Fyn. Measurements of 1H relaxation and interactions between H2O solvent and exchangeable protons are given, as well as a method for increasing the effective longitudinal relaxation of solvent exchangeable proton resonances. The long-range isotope shifts are measured, for 1H and 15N, which arise due to perdeuteration. Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time series of 2D NOESY datasets for some resonances. The agreement between experiment and simulation suggest that, with this 1H dilute sample, relatively long mixing times (up to 1.2 s) can be used to detect specific dipolar interactions between amide protons up to about 7A apart. A set of 155 inter-amide NOEs and 7 side chain NOEs were thus identified in a series of 3D HSQC-NOESY-HSQC experiments. These data, alone and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR. These results are compared to the available X-ray and NMR structures of the Fyn SH3 domain.

Solution structure of SpoIIAA, a phosphorylatable component of the system that regulates transcription factor sigmaF of Bacillus subtilis.

The establishment of differential gene expression in sporulating Bacillus subtilis involves four protein components, one of which, SpoIIAA, undergoes phosphorylation and dephosphorylation. We have used NMR spectroscopy to determine the solution structure of the nonphosphorylated form of SpoIIAA. The structure shows a fold consisting of a four-stranded beta-sheet and four alpha-helices. Knowledge of the structure helps to account for the phenotype of several strains of B. subtilis that carry known spoIIAA mutations and should facilitate investigations of the conformational consequences of phosphorylation.

Evidence that C1q binds specifically to CH2-like immunoglobulin gamma motifs present in the autoantigen calreticulin and interferes with complement activation.

Calreticulin (CRT) is located predominantly in the endoplasmic reticulum (ER) of cells, where it functions as a quality control controller of protein folding. However, CRT is also a prevalent autoantigen in patients with systemic lupus erythematosus (SLE), where its release from the cell may arise as a results of dysfunctional apoptosis and inefficient removal of ER vesicles, which are an abundant source of CRT and other autoantigens. Indicative of this is the presence of autoantibodies against CRT in the sera of 40-60% of all SLE patients. Once released into the circulation, CRT might bind directly to C1q and we have suggested that this association may result in a defect in C1q-mediated clearance of antigen-antibody complexes. It has been previously shown that CRT under physiological salt conditions binds to the globular head of C1q. It is known that the globular head region of C1q binds to the CH2 domain in the Fc portion of immunoglobulin gamma (IgG). The N-terminal half of CRT contains a number of short regions of 7-10 amino acids that show sequence similarity to the putative C1q binding region in the CH2 domain of IgG. By use of a series of 92 overlapping CRT synthetic peptides, a number of C1q binding sites on the CRT molecule have been identified, including several containing a CH2-like motif similar to the ExKxKx C1q binding motif found in the CH2 domain of IgG. A number of these peptides were shown to inhibit binding of C1q to IgG and reduce binding of native CRT to C1q. Moreover, several of the peptides were capable of inhibiting the classical pathway of complement activation. These studies have identified specific binding sites on the CRT molecule for C1q and lend support to the hypothesis that interaction of CRT with C1q may interfere with the ability of C1q to associate with immune complexes in autoimmune-related disorders.

Solvent structure at a hydrophobic protein surface.

The impact of an extensive, uniform and hydrophobic protein surface on the behavior of the surrounding solvent is investigated. In particular, focus is placed on the possible enhancement of the structure of water at the interface, one model for the hydrophobic effect. Solvent residence times and radial distribution functions are analyzed around three types of atomic sites (methyl, polar, and positively charged sites) in 1 ns molecular dynamics simulations of the alpha-helical polypeptide SP-C in water, in methanol and in chloroform. For comparison, water residence times at positively and negatively charged sites are obtained from a simulation of a highly charged alpha-helical polypeptide from the protein titin in water. In the simulations the structure of water is not enhanced at the hydrophobic protein surface, but instead is disrupted and devoid of positional correlation beyond the first solvation sphere. Comparing solvents of different polarity, no clear trend toward the most polar solvent being more ordered is found. In addition, comparison of the water residence times at nonpolar, polar, positively charged, or negatively charged sites on the surface of SP-C or titin does not reveal pronounced or definite differences. It is shown, however, that the local environment may considerably affect solvent residence times. The implications of this work for the interpretation of the hydrophobic effect are discussed.

Occurrence of Listeria and listeriosis in Hungary.

Listeriosis is a rare human disease in Hungary. The number of cases is slowly increasing. Only sporadic events have been observed but the occurrence of epidemic listeriosis may be supposed. The Listeria monocytogenes (in abbreviation: L. m.) transmitter role of food in human infections has not yet been verified. The epidemiological character of animal listeriosis is different. Healthy carriers can be found among both humans and animals. Foodstuffs of animal as well as plant origin may be contaminated with Listeria. When the processing technology and/or hygienic conditions are not satisfactory, these microorganisms can be detected in food factories and in final products of the food industry.

[Neurofibromatosis (von Recklinghausen disease) and carcinoid tumor in Vater's ampulla]. / Neurofibromatosis (von Recklinghausen-betegség) és carcinoid tumor a Vater-papillában.

The localization of the carcinoid tumors of the gastrointestinal tract--as a part of the APUD system--is extremely seldom at the region of ampulla of Vater. A case of congenital neurofibromatosis is reported in a 49-year-old male patient, with a carcinoid tumor of the ampulla of Vater in the background of long-standing, atypical biliary complaints. Periampullary neoplasm of neural-crest origin are very rare, up to this time 73 cases of Vater papilla carcinoid tumors were described. Regarding the development of VP carcinoid in neurofibromatosis, our patient is the 21st reported case in the world literature, and the first one in Hungary. The authors review the literature, giving special attention to the risk of periampullary neoplasms in von Recklinghausen's disease, the need of accurate diagnosis and the correctly selected operative intervention.

The effect of environment on the stability of an integral membrane helix: molecular dynamics simulations of surfactant protein C in chloroform, methanol and water.

A series of three molecular dynamics simulations at 300 K in explicit solvent environments of chloroform, methanol and water has been performed on the pulmonary surfactant lipoprotein, SP-C, comprising several consecutive valine residues in order to investigate the stability of the alpha-helical conformation. Two additional simulations were performed on truncated SP-C with a five-residue N-terminal deletion at 300 K and 500 K in water, the high temperature run in order to increase the rate of peptide denaturation. Indications of destabilization appear in chloroform during 1 ns while the SP-C alpha-helix is remarkably stable during 1 ns in methanol and water. In particular the polyvalyl part comprising residues Val15 to Val21 remains intact even at elevated temperature, and the valines do not disrupt the alpha-helical conformation. The valyl-rotamer sampling is partly restricted. Unfolding takes place successively along the primary sequence starting from the C-terminal end. Factors affecting polypeptide stability in molecular dynamics simulations are addressed. The intrinsic helix-forming tendency of valine residues and its dependence on the sequence context, and the role of the solvent environment in stabilizing or destabilizing an alpha-helical fold, are discussed.

Investigation of protein unfolding and stability by computer simulation.

Structural, dynamic and energetic properties of proteins in solution can be studied in atomic detail by molecular dynamics computer simulation. Protein unfolding can be caused by a variety of driving forces induced in different ways: increased temperature or pressure, change of solvent composition, or protein amino acid mutation. The stability and unfolding of four different proteins (bovine pancreatic trypsin inhibitor, hen egg white lysozyme, the surfactant protein C and the DNA-binding domain of the 434 repressor) have been studied by applying the afore-mentioned driving forces and also to some artificial forces. The results give a picture of protein (in)stability and possible unfolding pathways, and are compared to experimental data where possible.

Refined solution structure of the glucocorticoid receptor DNA-binding domain.

A refined solution structure of the glucocorticoid receptor DNA-binding domain (GR DBD) has been determined using two- and three-dimensional nuclear magnetic resonance (NMR) spectroscopy on an 15N-labeled GR DBD fragment in conjunction with distance geometry and simulated annealing calculations. Thirty structures of the fragment C440-R510 of the rat GR were calculated based on 906 distance constraints obtained from NOE intensities (168 intraresidue and 738 interresidue NOEs) and 43 dihedral constraints. Average atomic root mean square (rms) differences between the 24 best structures and their geometric average are 0.70 A for backbone atoms and 1.44 A for all heavy atoms. Several regions that were not well defined in a previous NMR structure determination of a similar protein fragment [Härd, T., Kellenbach, E., Boelens, R., Maler, B.A., Dahlman, K., Freedman, L.P., Carlstedt-Duke, J., Yamamoto, K.R., Gustafsson, J.-A., & Kaptein, R. (1990b) Science 249, 157-160] are now well-defined. The refined structure of the uncomplexed GR DBD is very similar to the crystal structure of GR DBD in a sequence specific DNA complex [Luisi, B. F., Xu, W. X., Otwinowski, Z., Freeman, L. P., Yamamoto, K. R., & Sigler, P. B. (1991) Nature 352, 497-505], in particular with regard to the presence and relative positions of secondary structure elements. The backbone atom rms difference between the average NMR solution structure and the crystal structure of the DNA-complexed GR DBD is 1.8 A. The most pronounced differences between the free and DNA-complexed states are found within the fragment C476-C482 in the second zinc-coordinating domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Backbone dynamics of the glucocorticoid receptor DNA-binding domain.

The extent of rapid (picosecond) backbone motions within the glucocorticoid receptor DNA-binding domain (GR DBD) has been investigated using proton-detected heteronuclear NMR spectroscopy on uniformly 15N-labeled protein fragments containing the GR DBD. Sequence-specific 15N resonance assignments, based on two- and three-dimensional heteronuclear NMR spectra, are reported for 65 of 69 backbone amides within the segment C440-A509 of the rat GR in a protein fragment containing a total of 82 residues (MW = 9200). Individual backbone 15N spin-lattice relaxation times (T1), rotating-frame spin-lattice relaxation times (T1 rho), and steady-state (1H)-15N nuclear Overhauser effects (NOEs) have been measured at 11.74 T for a majority of the backbone amide nitrogens within the segment C440-N506. T1 relaxation times and NOEs are interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) characterizing internal motions in each backbone amide using an optimized value for the correlation time for isotropic rotational motions of the protein (tau R = 6.3 ns). Average S2 order parameters are found to be similar (approximately 0.86 +/- 0.07) for various functional domains of the DBD. Qualitative inspection as well as quantitative analysis of the relaxation and NOE data suggests that the picosecond flexibility of the DBD backbone is limited and uniform over the entire protein, with the possible exception of residues S448-H451 of the first zinc domain and a few residues for which relaxation and NOE parameters were not obtained. in particular, we find no evidence for extensive rapid backbone motions within the second zinc domain. Our results therefore suggest that the second zinc domain is not disordered in the uncomplexed state of DBD, although the possibility of slowly exchanging (ordered) conformational states cannot be excluded in the present analysis.

Model examination of selective media for isolation of Listeria strains.

During the Tenth International Symposium on Listeriosis (Pécs, Hungary, 1988) the Working Party on Culture Media of IUMS-ICFMH suggested comparative examination of nine enrichment broths and nine solid selective media. On the basis of this proposal the following media were studied: LiCl-phenylethanol-moxalactam agar (LPM), polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol agar (PALCAM) No. 1 (home made) and No. 2 (Merck), acriflavine-ceftazidime agar (AC), Oxford agar, tripaflavine-nalidixic acid serum agar (TNSA) and Forray's agar. The study was performed as described in "Testing methods for use in quality assurance of culture media". Oxford agar proved to be the best medium. LPM, AC and Forray's agars were somewhat more inhibitory than Oxford medium. In productivity TNSA and PALCAM media were weakest but the latter one was more selective. When 43 sausage samples were enriched in UVM broths and subcultured on the above mentioned media the number of positive samples was the same on Oxford, LPM, AC and TNSA agars but it was lower on PALCAM agar No. 1. When 103 milk samples were subcultured on TNSA and PALCAM agar No. 2, the number of positive samples was the same.

A nationwide evaluation of multiple congenital abnormalities in Hungary.

A population-based study of 7,049 index patients with multiple congenital abnormalities (MCA) born in Hungary during 1973-1982 was organized by the Hungarian Center for Congenital Anomaly Control. All clinically recognized syndromes and associations which were submitted (2,049) were accepted without any further follow-up. New or supplementary information was requested in the case of unspecified MCA (320). A copy of detailed necropsy records was requested from pathologists in lethal cases (2,022). Following these steps, apparent but not true instances of MCA were excluded (399), and an attempt was made to assign as many of the remainder as possible in 17 well-delineated MCA entities (900). The living index patients with severe MCA were referred where possible to the regional centers for evaluation (864). One hundred and seventy entities were identified, and seven cases were excluded as not representing MCA. In the so-called 3,393 unidentified cases for which no diagnosis was possible, the component abnormalities were tabulated according to their number. The final count was 6,643 cases with MCA, which is equivalent to a birth prevalence of 4.0 per 1,000 total births, and to 10% of recorded cases with congenital anomalies. As a result of this program the proportion of recognized syndromes and associations among children with MCA increased from 29% to 47%. The accuracy of diagnoses has improved, e.g., the occurrence of unspecified cases decreased from 4.5% to 2%. As a result of this study, the number of chromosomal (1,700), Mendelian (557), and teratogenic (104) syndromes and associations (758) was considerably greater than the initial notifications indicated.

Sensitivity of 125J-fibrinogen uptake test for diagnosis of established venous thrombosis in heparinized patients.

The sensitivity of the radiofibrinogen test was investigated to determine the accuracy of the test in diagnosis of venographically proven venous thrombosis in 70 heparinized patients. 125J-fibrinogen was administered between 2 h and 4 days (mean: 1.5 days) after initiating heparin therapy. There were two criteria for a positive test: if the difference between the counts for adjacent points on the same leg or equivalent sites on opposite legs was at least 15%; and/or if there were three adjacent points each with counts greater than 5% that of the same three points on the other leg. The fibrinogen uptake test was positive at the first examination in 67 of 70 patients, giving a sensitivity of 96%. The respective frequencies of truly abnormal results for the 3 X 5% and the 1 X 15% criteria were 94% and 71%. As far as localization and extension of thrombosis were concerned, the fibrinogen uptake test agreed with phlebography in 73% of the cases, when the counts in the groin and the upper third of the thigh were ignored. The 125J-fibrinogen uptake test is an accurate method for detecting established deep leg vein thrombosis even in anticoagulated patients.