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The structure of dense grafted polyelectrolyte layers has been studied with a combination of neutron reflectivity and infrared spectroscopy techniques. The polyelectrolyte brushes were made of poly(styrene sulfonate) neutralized by different counterions. Small counterions are distributed throughout the brush in order to ensure a highly local electroneutrality. In addition, they can be readily exchanged with other small ions. On the other hand, macromolecular counterions (as well as some proteins) are irreversibly trapped by the brush, but are located outside the grafted layer and cannot reach the surface.

Stanniocalcin (STC) is a hormone that was originally identified in fish, where it inhibits calcium uptake by the gills and gut and stimulates phosphate adsorption by the kidney. Recently, two mammalian homologues of stanniocalcin were identified. The first (STC1) shows 61% identity to the fish stanniocalcins and appears to have a function similar to that of the fish stanniocalcins. The second homologue (STC2) is 30-38% identical to the fish stanniocalcins, and is characterized by unique cysteine and histidine motifs that are not found in the other stanniocalcins. We purified both the native hamster and recombinant human STC2 proteins and obtained a partial amino acid sequence of the hamster protein. Both proteins behave as a disulfide bonded homodimer, which undergoes post-translational modification(s). The STC2 gene was localized to human chromosome 5q35. Northern blot analysis revealed that the primary site of human STC2 production is the pancreas, and immunostaining localized the STC2 protein to a subpopulation of cells in the islet. Double immunostaining for STC2 and either insulin or glucagon revealed that STC2 protein is found in the alpha cells, but not the beta cells. We speculate that STC2 may play a role in glucose homeostasis.

We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD65) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD65 at levels approaching 0.5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD65 was purified to greater than 90% purity.

Thrombopoietin (TPO) is a hematopoietic factor involved in the regulation of megakaryocytopoiesis. Full length recombinant human TPO (332 residues) has been expressed in BHK cells and purified to homogeneity using conventional means. Peptide, disulfide, and glycosylation mapping of human TPO from residues 1 to 246 has been carried out using liquid chromatography-electrospray mass spectrometry (LC-ESMS). A modification of the ramped orifice method of Carr and co-workers [Carr et al. (1993) Protein Sci. 2, 183-196] is employed, providing additional information for assignment of the LC-ESMS chromatograms. With the modification, b- and y-series peptide ions are produced via front-end CID which confirms the mass-based assignments. The results of our analysis of TPO indicate that the amino acid sequence of TPO 1-246 is as expected from the transfected cDNA with complete cleavage of the signal peptide. Two unique disulfides are formed between the four cysteines in the cytokine domain of TPO: Cys7-Cys151 and Cys29-Cys85. The glycosylation map indicates the position, occupancy, and structures of the N- and O-glycans in TPO 1-246. In addition, site specific structural characterization of the PNGase F-liberated N-glycans has been performed following purification by high-pH anionic exchange chromatography with pulsed amperometric detection (HPAEC-PAD); the results corroborate the LC-ESMS data. The N-glycans are of the complex type with the core-fucosylated disialylated biantennary and trisialylated triantennary structures predominating. The O-glycans are of the mucin type with the monosialylated and disialylated GalGalNAc-S/T structures predominating. Furthermore, we propose that the C-terminal domain of TPO be further divided into two domains on the basis of sequence homology among the cloned sequences and glycosylation/structural features: an N-glycan domain (154-246) and an O-glycan domain (247-332).

The study comprised 236 pigs selected for resistance or susceptibility to oedema disease. The susceptibility to colonization of the small intestine by an Escherichia coli strain causing oedema disease was determined: (1) by monitoring faecal excretion of weaned pigs orally inoculated with E. coli strain O139:K12(B):H1:F18ab serotype; and (2) by an in vitro adhesion assay using an F18ab positive E. coli strain and small intestinal enterocyte preparations. Susceptibility to adhesion by these bacteria was shown to be controlled by a dominant (B) allele of the ECF18R locus and resistance by the alternative recessive allele (b). Pigs were typed for 14 blood group systems, 11 biochemical polymorphisms and the polymorphism at nucleotide 1843 of the RYR1 locus. Linkage was demonstrated between the locus for F18 E. coli receptors and the loci S, RYR1, GPI, EAH, A1BG and PGD (Z > 20). The most likely gene orders are: S-ECF18R-RYR1-GPI-PGD or GPI-RYR1-ECF18R-S-PGD. The recombination frequencies between ECF18R-S and ECF18R-RYR1 were estimated to be theta = 0.5% and 3.1%, respectively.

Two studies were conducted to evaluate high-quality tall fescue hay as a supplement to beef cattle fed low-quality roughages. In Exp. 1, 15 ruminally cannulated Hereford x Angus steers (average weight 390 kg) were blocked by weight and assigned randomly to one of three treatments: 1) tall fescue straw, no supplement; 2) tall fescue straw plus tall fescue hay supplement; 3) tall fescue straw plus alfalfa hay supplement. The 28-d digestion study consisted of 14 d of adaption, 6 d of intake data, and 6 d of collection of feces, respectively, with a 1-d ruminal sampling (d 27) and ruminal evacuations (d 28). In Exp. 2, 90 gestating Hereford x Angus cows were stratified by age and body condition and, within stratum, assigned randomly to three replications of the same treatments as described for Exp. 1. In both studies, a basal diet of tall fescue straw was fed with ad libitum access, alfalfa hay was fed at .4% BW, and tall fescue hay was fed at a level isonitrogenous with the alfalfa hay (.61% BW). In Exp. 1, DMI was at least 13% greater (P < .01) for supplemented steers than for nonsupplemented steers and was 12% greater (P < .10) for steers receiving supplemental tall fescue hay than for alfalfa hay-supplemented steers. Digestibility of DM was greater for supplemented steers than for nonsupplemented steers (P < .05) and, between supplement treatments, greater for tall fescue hay-supplemented steers than for alfalfa hay-supplemented steers (P < .10). Ruminal ammonia values peaked at 3 h after feeding and were higher for steers fed supplement treatments than for those fed the control treatment from just before feeding through 6 h after feeding (P < .10). In Exp. 2, supplemented cows gained more BW than nonsupplemented cows (P < .01), and the tall fescue hay-supplemented cows gained more BW (P < .10) than cows supplemented with alfalfa hay. Likewise, supplemented cows lost less condition (P < .01) than their nonsupplemented counterparts during the 84-d supplementation period, and cows receiving tall fescue hay supplement tended (P = .23) to lose less condition than cows receiving alfalfa hay supplement. No differences in calf growth were noted among treatment groups (P < .10). In conclusion, supplementation of high-quality tall fescue hay to cows fed low-quality forage diets seems to result in performance that is similar to or better than that of cows receiving alfalfa hay supplements when fed on an isonitrogenous basis.

The relatedness of the fimbriae produced by eight E. coli strains including type strains with F107 fimbriae, 2134P pili and colonization factor 8813 (preliminary F18), was examined. These strains had been isolated principally from pigs which were affected with postweaning diarrhoea or with oedema disease. The fimbriae were analyzed by means of electron microscopy, slide agglutination, immunofluorescence, immunogold labelling, immuno-diffusion, immunoelectrophoresis and western blot, molecular genetic techniques, and in vitro adhesion. The fimbriae of all the strains were long flexible filaments with a diameter not larger than 4.6 nm showing a zig-zag pattern. Results obtained by the serological techniques confirmed that the fimbriae possessed a common antigenic determinant designated 'a' in addition to a variant-specific determinant designated 'b' or 'c'. Immunoelectron microscopy demonstrated that the determinants 'a' and 'b' or 'a' and 'c' were localized along the same fimbrium. In immunoelectrophoresis, fimbrial extracts of selected strains yielded a single precipitation line towards the cathode. One single major subunit of approximately 15 kDa was recognised in western blots by antisera against the common antigenic determinant and the variant specific determinants. All strains possessed sequences related to gene fedA, coding for the major subunit of fimbriae F107. Two types of fedA-related subunit genes were differentiated, corresponding to the 'ab' and 'ac' types of fimbriae as defined by serological methods. The results demonstrated that F107 fimbriae, 2134P pili and colonization factor 8813 are related, and that two serological variants can be distinguished. We propose designations F18ab (for F107), and F18ac (for 2134P and 8813) in analogy to the nomenclature of F4 fimbriae.

Objective: To determine whether decision analysis is applicable to routine management of suspected pulmonary embolism in an emergency care setting., Design: Controlled feasibility trial., Setting: Emergency center of a university hospital., Patients: Outpatients (n = 84) admitted with clinical and scintigraphic evidence of pulmonary embolism., Interventions: Patients were treated either with the usual clinical work-up for pulmonary embolism (control group) or using a decision analysis model with three options: no action: angiography followed by treatment if positive; treatment without angiography., Results: All six senior residents in the decision analysis group agreed to fully participate for the 16 months of the study. Summarizing the decision analysis model in a graph was critical to obtain acceptance from all the physicians. Decision analysis (n = 43) and control (n = 41) patients underwent similar numbers of angiographies. However, angiographies for patients who had intermediate clinical probabilities of pulmonary embolism, between 25 and 75%, were more frequent in the decision analysis group (9/13 = 69%) than in the control group (7/20 = 35%). Agreement between clinical probability and lung-scan result was stronger in the decision analysis group., Conclusions: Decision analysis was successfully used to manage all patients suspected of having pulmonary embolism admitted to an emergency center during the 16-month trial. There was no insuperable obstacle to acceptance of clinical decision analysis by the physicians. Decision analysis may have resulted in a better discrimination between low and intermediate clinical probabilities of pulmonary embolism.

Expression of the fimbrial antigen F107 of porcine E. coli strains on agar plates was achieved by microaerobic cultivation. For part of the strains of some types, addition of alizarin yellow and eosin to the agar medium proved to be necessary. Some of these strains reacted distinctly positive only when the small colonies growing between the larger ones on alizarin-yellow agar were tested. The fimbrial antigen of the Swiss strain 107/86 was provisionally designated the F107ab variant and that of the Hungarian strain 2134P and the Czech strain 8813, the F107ac variant. The F107 genetic determinants were found to be often linked with those encoding haemolysin production and are frequently carried by plasmids.

Atherosclerotic carotid artery disease (ACAD) as a cause of objective-pulsatile tinnitus has not been adequately addressed in the literature. This report presents the authors' experience in 12 patients with pulsatile tinnitus arising from ACAD. Ipsilateral carotid bruit was detected in all patients and objective tinnitus was present in eight cases. The intensity of the carotid bruit was louder than the objective tinnitus in all patients. Carotid artery stenosis was confirmed in all patients with noninvasive vascular flow mapping. Atherosclerotic carotid artery disease should be highly suspected in patients with pulsatile-objective tinnitus who are older than 50 years and have associated cardiovascular risk factors. Pulsatile tinnitus in these patients can be the first manifestation of ACAD. Duplex ultrasound study of the carotid arteries can confirm the diagnosis. Consultation with a vascular surgeon is essential in selected cases.

Two experiments were conducted to evaluate digestion, performance, and physiological responses to Acremonium coenophialum-infected tall fescue straw offered to vary ergovaline concentrations. In Exp. 1, 16 Hereford x Angus ruminally cannulated steers (370 +/- 12 kg BW, mean +/- SE) were blocked by age and BW and, within block, randomly assigned to one of four treatments containing graded concentrations of ergovaline in the diet: 1) 0 ppb, 2) 158 ppb, 3) 317 ppb, and 4) 475 ppb. Alkaloid concentrations were produced using various mixtures of two varieties of tall fescue straw that had similar genetic and phenotypic characteristics but differed in degree of endophyte incidence and associated concentrations of alkaloids. In the 36-d digestion study, feed intake, apparent DMD, and total tract NDF digestion were not influenced by alkaloid concentration (P > .10). Differences were observed with digesta kinetics; specifically indigestible ADF (IADF) fill and outflow decreased linearly with increasing alkaloid concentration (P < .10). These differences, however, may be attributed to differences in IADF concentration of the diets. Physiological variables (heart rates, respiration rates, and rectal, ear, and tailhead skin surface temperatures) were not influenced by alkaloid concentration (P > .10). Concentration of serum prolactin measured weekly tended (P < .10) to decrease with increasing alkaloid concentration of the diet. At the conclusion of the study, each steer was injected with 100 micrograms of thyrotropin-releasing hormone (TRH). All steers responded to the TRH challenge (P < .10); however, responses did not differ (P > .10) between alkaloid concentrations of the basal diet (P > .10).(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-link formation, within the duplex, by oligodeoxynucleotides containing a 5-[omega-(omega-haloacylamido)alkyl]-2'-deoxyuridine to a complementary oligodeoxynucleotide was investigated under conditions approximating the physiologic environment. The site and extent of crosslinking to the target strand were determined for several electrophilic haloacylamidoalkyl structures. The regiospecificity of alkylation was primarily determined by the length of the electrophilic haloacylamidoalkyl group, while the extent of reaction was dependent upon both the structure of the acylamidoalkyl group and the reactivity of the electrophile. Cross-linking was additionally modulated by the sequence of the duplex in the vicinity of the alkylation site. The exact placement of the electrophile adjacent to the targeted nucleophile, an N-7 group on a specific guanine base in the target strand, was the most important factor in determining the rate of reaction. With the optimal haloacylamidoalkyl structure and duplex sequence, the most rapid rate obtained was t1/2 = 1.3 h at 37 degrees C.

This review describes the mechanism of intestinal colonization by enterotoxigenic and enterotoxaemic Escherichia (E.) coli in swine. Colonization depends on specific binding between adhesive fimbriae and receptors on the enterocytes. Special focus is given on the occurrence of specific intestinal receptors for the fimbrial antigens F4 (K88), F5 (K99), F6 (987P) and F41. The demonstration of these receptors allows the identification of susceptible and resistant pigs. Furthermore unclassified adhesive fimbriae of E. coli isolated from pigs with postweaning diarrhea and edema disease are described. Finally the present knowledge is discussed in context with protection of animals against infections.

A novel dATP analogue, 3-[5-[(N-biotinyl-6- amiocaproyl)amino]pentyl]-1-(2-deoxy-beta-D-erythro-pentofuranosyl )-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5'-triphosphate (9, bio-13-dAPPTP), which is modified at the 3-position with a flexible linker arm bearing a terminal biotin moiety, has been synthesized. This nucleotide is readily incorporated into DNA probes by nick translation. These probes hybridize to complementary targets as well as probes labeled with bio-dUTP, as judged by slot blot. When incorporated into oligonucleotides, they do not cause the loss of hybridization efficiency that an N-6-substituted adenine nucleotide does when incorporated into the same sites in the oligonucleotide.

Two strains of E. coli O139:K12 (B):H1 were compared in vitro and in the intestinal environment. Both strains colonized the small intestines of experimentally inoculated pigs and exhibited in vivo a similar relationship to the microvillus border as enterotoxigenic E. coli (ETEC). Strain 107/86 grown on blood agar expressed numerous long flexible non-haemagglutinating fimbriae which were antigenically distinct from the known fimbriae of porcine ETEC. It adhered in vitro to porcine enterocyte brush border fragments. Strain 124/76 grown on blood agar was devoid of fimbriae and did not adhere to brush border fragments. However, fimbriae morphologically and antigenically indistinguishable from those of strain 107/86 were detected in the intestinal environment by direct immunofluorescence and by immuno electron microscopy.

A series of 4,4-disubstituted tetrahydro- and 4,4-disubstituted hexahydro-3H-pyrido[1,2-c]pyrimidin-3-ones (4 and 5, respectively) were prepared from 2-aryl-2-(2-piperidinyl)-4-[N,N-bis (1-methylethyl)amino] butanamides (2). Individual racemates of the piperidinyl amides 2 were converted to pure racemic diaza bicyclic compounds that were evaluated for antiarrhythmic activity in the Harris dog model and anticholinergic activity in a muscarinic receptor binding assay. Selected compounds were subsequently evaluated for hemodynamic effects in anesthetized dogs where blood pressure depression and negative inotropic activity were assessed. Of this group, 4a (R = CH3) and 5a (R = CH3) showed the most favorable pharmacological profiles; the former compound was chosen for toxicity testing over the latter due to its lack of noncompetitive inhibition of acetylcholine-induced contractions of guinea pig ileum segments. Clinical evaluation is now under way.

We report 2 families with an autosomal dominant syndrome of limb and hematologic abnormalities. The W Family was ascertained through AW, a 13-year-old girl, who was purportedly born without congenital anomalies and who was normal until 11 1/2 years when she developed acute lymphoblastic leukemia. She died 2 years later with CNS involvement. Her chromosomes, studied in the first weeks after diagnosis of the disease, were apparently normal. Her father had clinodactyly of both 5th fingers and was found to have panmyelocytopenia refractory to all treatment at 26 years. He died within a year of the onset of his anemia. This man's oldest brother was born with congenital malformations of the elbows and the hands and was healthy until 38 years when he was also found to have an "idopathic anemia" and panmyelocytopenia which was refractory to treatment except for transfusions; he died at age 42 years. Both men were initially thought to have the Fanconi anemia syndrome. Their mother died at 71 years of leukemia. DT, the propositus of the second family, was noted to have malformations of both hands at birth. At 21 months he had anemia for which he received transfusions. Family history reveals that several people on the paternal side have severe hand anomalies and a history of childhood anemia. The paternal grandfather died at age 51 of acute monocytic leukemia. Barring genetic heterogeneity, we think that the trait in the W and T families in the same. It is a pleiotropic autosomal dominant mutant which affects radial and ulnar development of the upper limbs and is associated with a relatively high risk of transient or permanent bone marrow arrest with or without leukemia. We propose the hypothesis that apparently increased risk of leukemia to Fanconi heterozygotes actually represents admixture with the WT syndrome and that Fanconi heterozygotes may not have an increased risk of leukemia.

Ten day embryonated eggs have been used for testing supernatants of pooled RA synovial tissues for the presence of the RA-active agent. A high thermal resistance of the agent was observed up to 40 min of heating both at 100 and 121 degrees C with complete loss of infectivity only after 60 min at 121 degrees C. The high thermal resistance at 121 degrees C suggested that the active agent does not depend on a double-stranded DNA or RNA. It probably has no protein component and has a minimal number of subunits for replication. The CsC1 ultracentrifugation identified a concentration of high infectivity at densities in the range of 1.440-1.490 g/ml in five experiments.

The cardiovascular profile of SC-36602, a new class 1A/1B antiarrhythmic agent, was compared to those of disopyramide, lidocaine, mexiletine, flecainide, encainide, lorcainide, and quinidine. These drugs were compared at their respective canine antiarrhythmic doses in a hemodynamic evaluation using anesthetized dogs. In another test using anesthetized dogs, the cardiovascular effects of cumulative doses of SC-36602 were assessed. The direct negative inotropic potential of each drug was also determined using isolated cat papillary muscles. In the hemodynamic study, SC-36602 and quinidine did not cause significant myocardial depression, measured as a decrease in the maximal first derivative of the left ventricular pressure. (Heart rate and diastolic filling pressure were not controlled in order to mimic clinical conditions). SC-36602, mexiletine, flecainide, and quinidine increased heart rate. SC-36602 and mexiletine caused a small increase in mean arterial pressure, whereas disopyramide and quinidine decreased it. Disopyramide was the only drug studied that increased left ventricular end-diastolic pressure. SC-36602, quinidine, and mexiletine increased index of cardiac effort. Disopyramide caused a decrease in the latter. Disopyramide, flecainide, encainide, and lorcainide lengthened the ECG intervals. Cumulative intravenous doses of SC-36602 up to 50 mg/kg produced significant decreases in mean arterial pressure, increases in heart rate, and increases in P-R and QRS intervals of the ECG relative to placebo-matched controls. SC-36602 had the least direct negative inotropic action of the drugs studied, as measured in isolated papillary muscles. These data suggest SC-36602, when compared to the other antiarrhythmic drugs studied, has the least amount of hemodynamic side-effects at its antiarrhythmic dose.