Identification of a Brevibacterium marker gene specific to poultry litter and development of a quantitative PCR assay.

AIM: To identify a DNA sequence specific to a bacterium found in poultry litter that was indicative of faecal contamination by poultry sources. METHODS AND RESULTS: Faecally contaminated poultry litter and soils were used as source material for the development of a quantitative polymerase chain reaction (qPCR) method targeting the 16S rRNA gene of a Brevibacterium sp. The identified sequence had 98% nucleotide identity to the 16S rRNA gene of Brevibacterium avium. The qPCR method was tested on 17 soiled litter samples; 40 chicken faecal samples; and 116 nontarget faecal samples from cattle, swine, ducks, geese, and human sewage collected across the United States. The 571-bp product was detected in 76% of poultry-associated samples, but not in 93% of faecal samples from other sources. Marker concentrations were 10(7) -10(9) gene copies per gram in soiled litter, up to 10(5) gene copies per gram in spread-site soils, and 10(7) gene copies per litre in field run-off water. Results were corroborated by a blinded study conducted by a second laboratory. CONCLUSION: The poultry-specific PCR product is a useful marker gene for assessing the impact of faecal contamination as a result of land-applied poultry litter. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the first quantitative, sensitive and specific microbial source tracking method for the detection of poultry litter contamination.

Thermolabile alkaline phosphatase from Northern shrimp (Pandalus borealis): protein and cDNA sequence analyses.

Sequence analysis of short fragments resulting from trypsin digestion of the thermolabile shrimp alkaline phosphatase (SAP) from Northern shrimp Pandalus borealis formed the basis for amplification of its encoding cDNA. The predicted protein sequence was recognized as containing the consensus alkaline phosphatase motif comprising the active site of this protein family. Protein sequence homology searches identified several eukaryote alkaline phosphatases with which the 475-amino acid SAP polypeptide revealed shares 45% amino acid sequence identity. Residues for potential metal binding seem to be conserved in these proteins. The predicted 54-kDa molecular mass of SAP is smaller than previously reported, but is consistent with our recent SDS-PAGE analysis of the native protein. Compared to its homologs, the shrimp enzyme has a surplus of negatively charged amino acids, while the relative number of prolines is lower and the frequency of aromatic residues is higher than in mesophilic counterparts.

Blood lead and erythrocyte protoporphyrin levels in Kazakhstan.

A pilot study was conducted to examine the extent of lead exposure and prevalence of iron deficiency in 3 major cities of Kazakhstan. Blood lead (B-Pb.) and erythrocyte protoporphyrin (ZnPP) levels of 475 children, age range 6 months to 7 yeas were measured. The mean B-Pb. levels in the different cities ranged from 4-7 micrograms/dl (minimum 1 to max 29 micrograms/dl) and similarly the mean ZnPP levels ranged from 26-32 micrograms/dl (minimum 12 and maximum 95 micrograms/dl), thus confirming low level lead poisoning of children at some sites. One to four year olds had greater than 10 micrograms/dl B-Pb in 18-27% cases compared with 3-7% cases in five to seven year olds. Prevalence of iron deficiency in 6 months to 4 year old children was the highest ranging from 28-86% compared with 4 to 15% in 4-7 year olds. However, there was remarkably low prevalence (4%) of iron deficiency in a group of 5-6 years olds. This study suggests that a targeted B-Pb and ZnPP monitoring together with an iron supplementation programme in the 3 cities of Kazakhstan is essential. Environmental education appears to have had a positive impact in lowering B-Pb at one site and should thus be expanded nationwide.

Haemostatic parameters related to lipids and adhesion molecules.

Several components of blood, e.g. lipids, coagulation and fibrinolytic factors, are thought to be important risk factors in cardiovascular diseases. The aim of this study was to correlate these risk factors and the soluble adhesion proteins, soluble P-selection (sP-selectin) and soluble vascular cell adhesion molecule (sVCAM-1), in healthy men and women as well as to unravel any effects of smoking. One hundred and forty-two fasting men (median age 36 years) including 39 smokers, and 124 women (median age 34 years) including 35 smokers, were tested between 0800 h and 1000 h. Fibrinogen correlated positively with white blood cells (WBC) (r = 0.25), prothrombin fragment 1.2 (F1.2) (r = 0.21), cholesterol (r = 0.27), beta-thromboglobulin (r = 0.29), Factor VII clotting activity (FVIIc) (r = 0.27) (all P < 0.0001), tissue plasminogen activator antigen (t-PAag) (r = 0.22, P < 0.0005), plasminogen activator inhibitor-1 antigen (PAI-1ag) (r= 0.20) and VCAM-1 (r= 0.19) (both P< 0.002). Cholesterol and triacylglycerol (TG) correlated positively with t-PA antigen (t-PAag) (r = 0.36 and r = 0.38), PAI-1 antigen (PAI-1ag) (r = 0.35 and r = 0.50), P-selectin (r = 0.26 and r = 0.27) (all P < 0.0001) and WBC (r = 0.17, P < 0.007 and r = 0.18, P < 0.004). Cholesterol correlated also with F1.2 (r = 0.29) and TG (r= 0.44) (P< 0.0001). In addition to cholesterol and TG, sP-selectin correlated postively with PAI-1ag (r= 0.39), t-PAag (r= 0.27) and WBC (r = 0.25) (all P < 0.0001). Comparing the various test parameters in men and women, it was found that women had significantly higher levels of F 1.2 and high-density lipoprotein-cholesterol than men, whereas men had higher levels of t-PAag, PAI-lag and P-selectin than women. Smoking was associated with a rise in several of the test parameters. It can be concluded that there are correlations between several risk factors. Of particular interest is the positive correlation between sP-selectin and a number of established risk factors of cardiovascular diseases.

Structure and proposed amino-acid sequence of a pepsin from atlantic cod (Gadus morhua).

The crystal structure of a pepsin from the gastric mucosa of Atlantic cod has been determined to 2.16 A resolution. Data were collected on orthorhombic crystals with cell dimensions a = 35.98, b = 75.40 and c = 108.10 A, on a FAST area-detector system. The phase problem was solved by the molecular-replacement method using porcine pepsin (PDB entry 5PEP) as a search model. The structure has been refined to a crystallographic R factor of 20.8% using all reflections between 8.0 and 2.16 A, without prior knowledge of the primary sequence. The resulting crystal structure is very similar to the porcine enzyme, consisting of two domains with predominantly beta-sheet structure in the same sequential positions as the enzyme from pig. In the course of the model building, 122 residues were substituted and two residues deleted from the starting model to give a polypeptide chain of 324 amino acids and a sequence identity of 57.7% with the pig pepsin. No carbohydrate residues were located. Sequence alignment with available aspartic proteinases, indicates that the fish enzyme seems to be more related to mammalian gastric pepsins than to the mammalian gastricsins and chymosins, lysosomal cathepsin D's and a pepsin from tuna fish. The amino-acid composition of the cod enzyme, however, is more in accordance with the cathepsin D's.

Effects of dietary marine oils and olive oil on fatty acid composition, platelet membrane fluidity, platelet responses, and serum lipids in healthy humans.

The influence of various dietary marine oils and olive oil on fatty acid composition of serum and platelets and effects on platelets and serum lipids were investigated as part of an extensive study of the effects of these oils on parameters associated with cardiovascular/thrombotic diseases. Healthy volunteers (266) consumed 15 mL/d of cod liver oil (CLO); whale blubber oil (refined or unrefined); mixtures of seal blubber oil and CLO; or olive oil/CLO for 12 wk. In the CLO, seal oil/CLO, and whale oil groups, serum levels of eicosapentaenoic acid (EPA) were increased. In platelets, EPA was increased in the CLO, seal/CLO, and olive oil/CLO groups. The localization of n-3 polyunsaturated fatty acids in the triacylglycerols did not seem to influence their absorption. Intake of oleic acid is poorly reflected in serum and platelets. No significant differences in triacylglycerols (TG), total cholesterol, or high density lipoprotein cholesterol were observed, even though TG were reduced in the CLO, CLO/seal oil, and whale oil groups. Mean platelet volume increased significantly in both whale oil groups and the CLO/olive oil group. Platelet count was significantly reduced in the refined whale oil group only. Lipopolysaccharide-stimulated blood tended to generate less thromboxane B2 in CLO, CLO/seal, and CLO/olive groups. The whale oils tended to reduce in vivo release of beta-thromboglobulin. In conclusion, intake of various marine oils causes changes in platelet membranes that are favorably antithrombotic. The combination of CLO and olive oil may produce better effects than these oils given separately. The changes in platelet function are directly associated with alterations of fatty acid composition in platelet membranes.

Effect of marine oils supplementation on coagulation and cellular activation in whole blood.

A study was performed to explore the effects of supplemental intake of various marine oils known to be part of the Eskimo diet. Healthy men and women (134) were randomly selected to consume 15 mL/d of oil from blubber of seal, cod liver, seal/cod liver, blubber of Minke whale, or no oil for ten weeks. Total cholesterol was unchanged in the oil groups, whereas high density lipoprotein cholesterol increased 7% in the seal/cod liver oil (CLO) group (P < 0.05) and 11% in the whale oil group (P < 0.005). Triacylglycerol was significantly reduced in the CLO group only. The concentration of prothrombin fragment 1 + 2 was reduced 25% (P < 0.05) after whale oil supplementation. No change in fibrinogen or factor VIIc was detected. Tumor necrosis factor generation in lipopolysaccharide (LPS)-stimulated blood was 30% reduced after whale oil (P < 0.05), but was unaffected by intake of seal or CLO. The LPS-induced tissue factor activity in monocytes was reduced to a significant degree only in the seal/CLO group (34%) and whale oil group (35%) (P < 0.05). The most dramatic change in thromboxane B2 in LPS-stimulated blood was seen after whale oil intake with 44% reduction (P < 0.01). Supplementation of a regular diet with a combination of seal oil and CLO and especially with whale oil seems to have beneficial effects on several products thought to be associated with cardiovascular and thrombotic diseases.

Comparison of myosin isoenzymes present in skeletal and cardiac muscles of the Arctic charr Salvelinus alpinus (L.). Sequential expression of different myosin heavy chains during development of the fast white skeletal muscle.

The expression of myosin isoforms and their subunit composition in the white skeletal body musculature of Arctic charr (Salvelinus alpinus) of different ages (from 77-day embryos until about 5 years old) was studied at the protein level by means of electrophoretic techniques. Myosin from the white muscle displayed three types of light chain during all the developmental stages examined: two myosin light chains type 1 (LC1F) differing in both apparent molecular mass and pI, one myosin light chain type 2 (LC2F) and one myosin light chain type 3 (LC3F). The fastest-migrating form of LC1F seemed to be predominant during the embryonic and eleutheroembryonic periods. The slowest-migrating form of LC1F was predominant in the 5-year-old fish. Between 1 year and 4 years, both types of LC1F were present in similar amounts. Cardiac as well as red muscle myosin from 3-year-old fish had two types of light chain. The myosin light chains from atria and ventriculi were indistinguishable by two-dimensional electrophoresis, but were different from the myosin light chains from red muscle. Neither the light chains from cardiac nor red muscle were coexpressed with the myosin light chains of white muscle at any of the developmental stages examined. Two myosin heavy chain bands were resolved by SDS/glycerol/polyacrylamide gel electrophoresis of the extract from embryos. One of the bands was present in minor amounts. The other, and most abundant, band comigrated with the only band found in the extracts of white muscle myosin from older fish. One-dimensional Staphylococcus aureus V8 protease peptide mapping of these bands revealed some differences during development of the white muscle tentatively interpreted as follows. The myosin heavy chain band present in minor amounts in the embryos may represent an early embryonic form that is replaced by a late embryonic or foetal form in the eleutheroembryos. The foetal myosin heavy chain appears to be present until the resorption of the yolk sack and beginning of the free-swimming stage. A new form of myosin heavy chain, termed neonatal and probably expressed around hatching, is present until about 1 year of age.(ABSTRACT TRUNCATED AT 400 WORDS)

Myosin isoforms in red and white muscles of some marine teleost fishes.

The myosin content from red and white muscles of three marine fish species, saithe (Pollachius virens. L.), haddock (Melanogrammus aeglefinus, L.), both members of the family Gadidae, and capeline (Mallotus villosus, M.) of the family Osmeridae, was analyzed electrophoretically. Analysis of the native myosin by electrophoresis under non-dissociating conditions revealed two isoforms in red muscles, and three or four in white muscles. The white muscles of the two closely related species had a similar pattern of isoforms. Myosin from the slow red muscles had two types of light chain, LC1S and LC2S, and myosin from the fast white muscles three, LC1F, LC2F, and LC3F. The pattern of light chains in both types of muscles was species-dependent. All the light chains from fish myosins were more acidic than those of the rat diaphragm used as standard. One main type of heavy chain was detected in each kind of muscle. In white muscles of saithe there was an extra band, present in minor amounts. The heavy chains from white muscle myosin had lower electrophoretic mobilities than those from red muscle, and the mobilities of all of them were intermediate between those of the heavy chains type IIa and I of rat diaphragm myosin. In our opinion, there are probably more isomyosins in fish muscles than those detected in the present work and their presence is obscured by comigration with the main types.

Intraspecific myosin light chain polymorphism in the white muscle of herring (Clupea harengus harengus, L.).

The myosin contained in white and red muscles of herring (Clupea harengus harengus) was purified, and its subunit composition analyzed by electrophoretic techniques. The only myosin isoform present in red muscles was made up of one type of heavy chain and two types of light chain. The native myosin from white muscles migrated as one wide band. Analysis of the extracts by SDS/glycerol/PAGE from white muscles revealed one main type of heavy chain. Light chains were identified by SDS-PAGE analysis of electrophoretically purified myosin, and two-dimensional electrophoresis of the extracts demonstrated differences in the light chain composition of white and red muscles. Using this methodology, light chain polymorphism was detected in white muscles among members of the same species.

Electrophoretic study of myosin isoforms in white muscles of some teleost fishes.

1. White skeletal muscle myosin of four marine teleost fish species (cod, blue whiting, Norway haddock, and spotted wolf-fish) was analyzed by native, SDS-PAGE, and 2-dimensional electrophoresis. 2. Four types of native myosin were present in cod, blue whiting and Norway haddock. The second fastest migrating form was predominant. 3. Myosin from spotted wolf-fish also resolved into four forms. The fastest migrating form was hardly noticeable. The other three were present in apparently similar amounts. 4. In the myosin from each species there were three types of light chains. The pattern of light chains was species specific. 5. Apparently, there was only one type of heavy chain in myosin from cod, Norway haddock and spotted wolf-fish. One preparation of cod showed an extra band of higher electrophoretic mobility than the main band. In blue whiting we found two bands present in approximately equal amounts.

Catalytic properties and chemical composition of pepsins from Atlantic cod (Gadus morhua).

1. Three pepsins were purified from the gastric mucosa of Atlantic cod (Gadus morhua). 2. The enzymes, called Pepsin I and Pepsin IIa and b, had isoelectric points 6.9, 4.0 and 4.1, respectively, and digested hemoglobin at a maximal rate at a pH of approximately 3. 3. They resembled bovine cathepsin D in being unable to digest the mammalian pepsin substrate N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine. 4. Specificity constants (kcat/Km) for the cod pepsins were lower than for porcine pepsin, and they expressed higher substrate affinity and physiological efficiency at pH 3.5 than at pH 2. 5. The cod pepsins are glycoproteins, and their amino acid composition resembles that of porcine cathepsin D more than that of porcine pepsin. 6. The N-terminal sequence of Atlantic cod pepsins is substantially different from that of porcine pepsin. This indicates a significant evolutionary gap between fish and mammalian pepsins.

Characterization of secretin-like immunoreactivity in pregnancy.

The plasma secretin-like immunoreactivity (SLI) in 23 healthy females was elevated in late pregnancy (34 +/- 3 pmol/l) as compared with 23 non-pregnant female controls (12 +/- 2 pmol/l; p less than 0.01). The plasma SLI in pregnancy eluted close to albumin on a Sephadex G-200 column, whereas 50-75% of the recovered SLI was displaced to the elution volume of free secretin when plasma was exposed to 6 mol/l urea. When 125I-labelled secretin was incubated with plasma in the absence of secretin antibodies, 40% of the intact label eluted in the void volume of a Sephadex G-50 Fine column in pregnancy, compared with only 18% in the nonpregnant state. The present study supports the notion that secretin circulates bound to plasma proteins and suggests that the protein binding of secretin is enhanced in late pregnancy, a feature common to several classical hormones.

Purification and characterization of two trypsin-like enzymes from the digestive tract of anchovy Engraulis encrasicholus.

1. Two trypsin-like enzymes, designated Trypsin A and B, were purified from the pyloric caeca and intestine of anchovy by (NH4)2SO4 fractionation, affinity chromatography (Benzamidine-Sepharose-6B) and ion exchange chromatography (DEAE-Sepharose). 2. Both trypsins catalyzed the hydrolysis of N-benzoyl-DL-arginine p-nitroanilide (BAPNA), p-tosyl-L-arginine methyl ester (TAME), casein and myofibrillar protein and they were inhibited by several well established trypsin-inhibitors. 3. The enzymes had mol. wts of 27,000 (Trypsin A) and 28,000 (Trypsin B). Their isoelectric points were about 4.9 (Trypsin A) and 4.6 (Trypsin B) and they had similar amino acid composition. 4. The enzymes had a pH optimum of 8-9 for the hydrolysis of BAPNA and of 9.5 for the digestion of casein and myofibrillar protein. Their activity and stability were affected by calcium ions. 5. Trypsins A and B resemble other fish trypsins in their mol. wt, pI, kinetic properties and the instability at low pH and they are similar to bovine trypsin in their dependence of Ca2+ for activity and stability.

Molecular forms of myeloperoxidase in human plasma.

A radioimmunoassay for myeloperoxidase was established with the use of affinity-purified anti-(human myeloperoxidase) immunoglobulins. By the use of ion-exchange followed by immunoaffinity chromatography a preparation of immunoreactive, catalytically active myeloperoxidase was obtained from fresh human plasma. In non-denaturing gel electrophoresis, the plasma preparation showed about four catalytically active components of mobility very similar to that of the granulocyte enzyme. SDS/polyacrylamide-gel electrophoresis combined with protein blotting showed that the two polypeptides of strongest antigenicity in the plasma preparation corresponded in Mr to the large and the small subunits of the granulocyte enzyme. In addition, the plasma preparation contained a higher-Mr immunoreactive polypeptide, possibly a precursor form of the enzyme, together with another of Mr similar to that of the large subunit of eosinophil peroxidase.

Further characterization of human eosinophil peroxidase.

The large and the small subunits (Mr 50 000 and 10 500 respectively) of human eosinophil peroxidase were isolated by gel filtration under reducing conditions. The subunits were very strongly associated but not apparently cross-linked by disulphide bridges. During storage, the large subunit tended to form aggregates, which required reduction to dissociate them. Amino acid analysis of the performic acid-treated large subunit showed the presence of 19 cysteic acid residues. The small subunit of eosinophil peroxidase had the same Mr value as the small subunit of myeloperoxidase. However, although these subunits have very similar amino acid compositions, they showed different patterns of peptide fragmentation after CNBr treatment. The carbohydrate of eosinophil peroxidase seemed associated exclusively with the large subunit and comprised mannose (4.5%, w/w) and N-acetylglucosamine (0.8%, w/w). The far-u.v.c.d. spectrum of the enzyme indicated the presence of relatively little ordered secondary structure.

Effect of intravenous and intraduodenal administration of Escherichia coli endotoxin on the porcine pancreas as evaluated by changes in the serum cationic trypsin-like immunoreactivity.

In six pigs intravenous administration of Escherichia coli endotoxin caused septic shock and a significant increase in serum cationic trypsin-like immunoreactivity (CTLI), with activation of cationic trypsinogen to trypsin and formation of complexes between cationic trypsin, on the one hand, and alpha 2-macroglobulin and alpha 1-antitrypsin, on the other, compatible with acute pancreatitis. In contrast, intraduodenal infusion of E. coli endotoxin to seven other pigs was without effect on the general circulation and on the serum CTLI.