Purification and characterization of endogenous digoxin-like immunoreactive factors in chicken blood

Studies have been performed to determine whether an endogenous material capable of binding to digoxin antibodies is present in the chicken plasma. In the blood of 12 chickens without feed control, endogenous digoxin-like immunoreactive factors (DLIF) binding of digoxin antibodies in enzyme immunoassays amounted to 866 / 302 pg digoxin equivalents/mL of plasma (mean +/- SEM). Immunoreactivity of DLIF increased to 1848***331 pg/mL with a double value of control after boiling and acid pretreating the plasma. The major purification steps employed in this report were gel filtration column chromatography, high performance liquid chromatography (HPLC) and isoelectric focusing (IEF). Using HPLC for the separation, at least 10 chicken DLIFs with different molecular weight (MW) have been found. The MW of the smallest is 300 daltons (Da) while the largest is 100 kDa. The value of the isoelectric point of the most abundant type of DLIF from untreated chicken plasma is 6.3 as determined by IEF. The partially purified DLIF inhibits Na+, K(+)-ATPase from a porcine cerebral cortex as well as three human red blood cell membrane preparations in a dose-response fashion.     

Serum digoxin-like immunoreactive factor in children and its relation to sodium metabolism

BACKGROUND: The definition of success and failure of a bariatric procedure should include weight loss as well as improvement in comorbidity and quality of life assessment. The Bariatric Analysis and Reporting Outcome System (BAROS), introduced by Oria and Moorhead in 1997, seems to provide a standard for comparison in the surgical treatment of morbid obesity. METHODS: 180 morbidly obese and super-obese patients, who underwent laparoscopic gastric banding (lap-band) at our institutions and had a follow-up >18 months (19-55 months) were evaluated with BAROS. RESULTS: The patients were divided into four outcome groups (failure, fair, good, and excellent) based on a scoring table that adds or subtracts points while evaluating three main areas: percentage of excess weight loss, changes in medical conditions, and assessment of quality of life. Points were deducted for complications and reoperative surgery. CONCLUSIONS: The BAROS outcome system has proved to be a useful instrument in evaluating midterm results in our series of lap-band patients.     

Atrial natriuretic factor and digoxin-like immunoreactive factor in diabetic patients: their interrelation and the influence of the autonomic nervous system

In diabetic patients, several factors contribute to volume expansion and have to be counteracted by humoral and neuronal feedback control systems. We investigated N-terminal proatrial natriuretic factor (ANF1-98) and digoxin-like immunoreactive factor (DLIF), which are two counteracting hormones, and their interrelationship, with additional consideration given to autonomic nervous function in diabetic patients. ANF1-98 and DLIF were measured in 64 diabetic patients. Autonomic nervous function was assessed using nine autonomic nervous function tests. The patients were subdivided into two groups, one with four or more (group 1) and one with less than four abnormal results in autonomic function tests (group 2). Compared with group 2, group 1 demonstrated detectable DLIF levels less often (17.2 vs. 45.7, P = 0.0195) and increased levels of ANF1-98 (mean +/- SEM: 850.0 +/- 108.8 vs. 554.8 +/- 45.9 pmol/L, P = 0.0099). However, the groups did not differ in blood pressure, daily sodium, and daily potassium excretion. The number of abnormal autonomic function tests correlated significantly with ANF1-98 (P = 0.0002). In patients with detectable DLIF, DLIF correlated with ANF1-98 (P = 0.0080). These results demonstrate close interactions between the autonomic nervous system and the two natriuretic hormones. In patients with autonomic nervous dysfunction, higher levels of ANF may possibly compensate for the lack of the natriuretic DLIF to counteract hypertension and chronic volume expansion.     

Digitalis-like and vasoconstrictor effects of endogenous digoxin-like factor(s) from the venom of Bufo marinus toad

Digitalis glycoside-like properties of the Bufo marinus toad crude venom and one of its constituents, bufalin, were studied in various assay systems. In concentrations 0.3-30 micrograms/ml crude venom increased the contractility of isolated electrically driven rat atria, constricted rat aortic rings, inhibited ouabain-sensitive Na+,K(+)-ATPase in rat erythrocytes and the Na+,K(+)-pump in rat aorta, and cross-reacted with antidigoxin antibody from the dissociation enhanced lanthanide fluoroimmunoassay (DELFIA). These effects were unaffected by adrenoceptor blockers and the 5-HT antagonist, deseril, but were blocked by antidigoxin antibody. Bufalin (10-30 microM) increased myocardial contractility and inhibited Na+,K(+)-ATPase in rat erythrocytes similarly to crude Bufo marinus venom. In rat aorta bufalin showed weak and delayed vasoconstrictor activity which was antagonized by 2 microM phentolamine, and had a biphasic effect on the Na+,K(+)-pump; 0.5-1.0 microM bufalin stimulated the pump, while higher concentrations inhibited its activity. Although the effects of bufalin were blocked by antidigoxin antibody, bufalin showed very low digoxin-like immunoreactivity in the DELFIA. These observations suggest that, in addition to bufalin, Bufo marinus venom contains at least one more digitalis-like steroid with significant intrinsic vasoconstrictor activity which, unlike bufalin, constricts the blood vessels acting directly via inhibition of the sodium pump in the vascular smooth muscle membrane.     

Comparison of four digoxin immunoassays with respect to interference from digoxin-like immunoreactive factors

OBJECTIVES: Comparison of a new monoclonal digoxin assay with three polyclonal digoxin assays for their cross-reactivity to digoxin-like immunoreactive factors (DLIF) and digoxin metabolites. DESIGN AND METHODS: Sixty-six nondigitalized patient samples from 5 different groups: neonates, women in 3rd trimester pregnancy, and patients with liver or renal diseases, or undergoing organ transplants, and 139 samples from digoxin-treated patients of 4 categories (hospital sick, liver, renal, and outpatients) were compared in 4 different digoxin assays: (a) ACS Digoxin (ACS) developed for the automated chemiluminescent Ciba Corning ACS 180 system, (b) Baxter Stratus (Stratus, a fluoroimmunoassay), (c) Ciba-Corning Magic (Magic, a radioimmunoassay), and (d) an in-house radioimmunoassay (RIA). The ACS and RIA were also compared for their cross-reactivity to four principal digoxin metabolites. RESULTS AND CONCLUSION: Among the nondigitalized specimens, no significant DLIF interference was found for all 4 assays among the pregnant women or liver and transplant patients. However, the neonates registered high DLIF interference with Magic and RIA, but none for ACS or Stratus. DLIF interference in renal samples was highest in the Magic assay and lowest in RIA. Among the specimens with digoxin, a higher number of discrepant samples were found from the sick patients than from outpatients. In 75% of such discrepant samples, the ACS result was less than other assay results, suggesting DLIF as the probable cause. The two assays differed most in their cross-reactivity to the deglycated metabolites, digoxigenin and its mono-digitoxoside.     

An investigation of immunoreactive substances in normal gingival tissue and periodontitis tissue

In order to study the effects of NO, endothelin (ET), substancep (SP) and caleiton gene-related paptide (CGRP) immunoreactive substances in chronic periodontitis tissue and the relationship with periodontitis, immunohistochemical and histochemical staining methods were used respectively to detect the expression of the above immunoreactive substances in 20 patients with chronic periodontitis. Quantitative analysis showed that NO and ET in periodontitis tissues were increased significantly (P < 0.01), particularly the content of ET, in comparison to normal tissue. Due to the increase of ET content, the bolld supply was diminished and influenced the periodontal tissue by causing tissue damage. This study suggests that NO and ET immunoreactive substances are closely associated with periodontitis and may play an important role in the disease. There were no marked changes on SP and CGRP.     

Calcium-dependent modulation of the agonist affinity of the mammalian olfactory cyclic nucleotide-gated channel by calmodulin and a novel endogenous factor

Capillary electrophoresis has been successfully employed to determine the level of drugs in a variety of pharmaceutical preparations. A large number of reports have shown agreement between CE results and HPLC data or with label claim. Currently the use of CE for main component assays constitutes 26% of the routine usage of CE within drug companies and is the most frequent application. The choice between adopting CE or HPLC for a particular application is very dependent upon the relative merits of each technique to the individual assay. Often, CE can have advantages in terms of reduced sample pretreatment, consumable costs, and analysis time. The ability to separate a wide range of solutes using a single set of operating conditions is a strong advantage of CE. This paper extensively reviews the literature reports of the use of CE for main peak assay and indicates the validation performance data achieved. The specific requirements relating to optimized accuracy and precision in CE assay are discussed in some detail. The use of an appropriate internal standard to improve performance for precision, accuracy, and linearity, and to reduce the impact of sample matrix effects, is experimentally shown by results from the analysis of levothyroxine samples. The applications are subdivided into those samples analyzed by free solution capillary electrophoresis (FSCE) at low or high pH or those separated by micellar electrokinetic capillary electrophoresis (MECC).     

Expression of initiation factor genes in mammalian cells

This review focuses on how cells establish the levels of initiation factors, within the broader context of determining levels of the translational machinery. Most initiation factor polypeptides are moderately abundant proteins with concentrations approaching those of ribosomes. eIF4A and eIF5A are more abundant than ribosomes, whereas eIF4F alpha and eIF2B are considerably less abundant than the other factors. The cloning of cDNAs generates hybridization probes for monitoring the levels and activities of factor mRNAs, and the cloning of their genes is just beginning to provide insight into promoter structures and regulation. Initiation factor gene expression appears to be coordinately regulated in many cases, and preferential synthesis is seen in mitogen-activated T-cells. The gene for eIF2 alpha has been best characterized, and mechanisms that provide for the coordinated synthesis of eIF2 subunits are emerging. Recombinant DNA methods also allow investigators to manipulate the levels of expression of specific factor genes by overexpression or antisense repression. Such approaches provide a means to investigate in vivo the mechanisms of action of the initiation factors and their roles in regulating translation rates.     

Distribution of endogenous tumour necrosis factor alpha in gliomas

AIMS: To determine the distribution and cellular origin of endogenous tumour necrosis factor alpha (TNF alpha) in the cellular components of human gliomas. METHODS: Frozen sections of 26 gliomas (four astrocytomas (As); two oligoastrocytomas (OA); one ansplastic astrocytoma (AA); one anaplastic oligoastrocytoma (AOA); 18 glioblastomas (GB)) were examined immunohistochemically using antihuman TNF alpha and anti-Leu-M5 (CD11c) antibodies. Additional studies with double immunohistocchemical procedures were performed with anti-glial fibrillary acidic protein and anti-neurofilament antibodies. RESULTS: Eighty per cent of the AA, AOA, and GB (16 of 20) had a positive reaction for TNF alpha, but only 17% of As and OA (one of six) were positive. Positive cells were seen in both the tumour tissue and adjacent brain tissues. TNF alpha protein was detected not only in the tumour cells but also in the endothelium of tumour vessels as well as reactive astrocytes and neurons. CONCLUSIONS: Endogenous TNF alpha is present in cells of various origins in glial tumours including tumour vessels; however, the role of TNF alpha may be different in different types of cells or altered microenvironment.     

Inositol 1,4,5-trisphosphate receptor expression in mammalian olfactory tissue

The interaction of recombinant ascorbate peroxidase (APX) with its physiological substrate, ascorbate, has been studied by electronic and NMR spectroscopies, and by phenylhydrazine-modification experiments. The binding interaction for the cyanide-bound derivative (APX-CN) is consistent with a 1:1 stoichiometry and is characterised by an equilibrium dissociation binding constant. Kd, of 11.6 +/- 0.4 microM (pH 7.002, mu = 0.10 M, 25.0 degrees C). Individual distances between the non-exchangeable substrate protons of APX-CN and the haem iron were determined by paramagnetic-relaxation NMR measurements, and the data indicate that the ascorbate binds 0.90-1.12 nm from the haem iron. The reaction of ferric APX with the suicide substrate phenylhydrazine yields predominantly (60%) a covalent haem adduct which is modified at the C20 carbon, indicating that substrate binding and oxidation is close to the exposed C20 position of the haem, as observed for other classical peroxidases. Molecular-modelling studies, using the NNM-derived distance restraints in conjunction with the crystal structure of the enzyme [Patterson, W. R. & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], are consistent with binding of the substrate close to the C20 position and a possible functional role for alanine 134 (proline in other class-III peroxidases) is implicated.     

Elevated tissue factor and tissue factor pathway inhibitor circulating levels in ischaemic heart disease patients

The number and arrangement of scutellar bristles on the thorax of Drosophila melanogaster is largely invariant in wild-type stocks. This character therefore appears to be buffered against changes in phenotype, and has previously been described as a canalized character. Mutations that do alter this phenotype increase the variability in bristle number and can reveal otherwise cryptic genetic differences at other loci. This phenomenon is examined and possible mechanisms contributing to stability of this developmental event are discussed, but the notion that the character is canalized is found not to be heuristic.     

Effect of a non-steroidal anti-inflammatory drug on tissue levels of immunoreactive prostaglandin E2, immunoreactive leukotriene, and pain after periodontal surgery

The aim of this study was to measure tissue levels of immunoreactive prostaglandin E2 (iPGE2), immunoreactive leukotriene B4 (iLTB4), and pain after periodontal surgery and to evaluate the effect of the non-steroidal anti-inflammatory drug (NSAID), ibuprofen, on these levels. Two contralateral quadrants in each of nine patients were selected to undergo separate surgical procedures, one with ibuprofen (800 mg 1 hour presurgery and 400 mg postsurgery) and one with a placebo. Intra-operatively, a custom-made microdialysis probe, with a 3,000 dalton molecular weight cut-off, was inserted beneath the soft tissue flap and a dialysate collected every 20 minutes for 4 hours after surgery. Pain perception was measured at the same time intervals using two pain scales. Dialysate samples were assayed using two enzyme immunoassays. Mean tissue levels of iPGE2 in the placebo group increased from 74 nM at 40 minutes to a peak of 261 nM at 200 minutes. Mean tissue levels of iLTB4 in the placebo group fluctuated between 0.2 and 0.6 nM. Pain levels in this group increased continuously with time, peaking at 4 hours. Mean tissue levels of iPGE2 in the ibuprofen group were significantly suppressed, exhibiting more than a 95% reduction. This was accompanied by a significant reduction in pain. Ibuprofen had no detectable effect on tissue levels of iLTB4. These data indicate that iPGE2 and iLTB4 are present at relatively high concentrations in the periodontal tissues after surgery. Since these concentrations exceed the Kd values for binding to their respective receptors, PGE2 and LTB4 may be associated with the development of postsurgical pain and inflammation. These data also indicate that ibuprofen can successfully inhibit iPGE2 production in the periodontal tissues and in this way may help reduce postoperative pain and inflammation.     

Eimeria coecicola Cheissin 1947: endogenous development in gut-associated lymphoid tissue

Coccidia-free rabbits were inoculated with different doses of a pure strain of Eimeria coecicola and samples of gut were taken at 80, 96, 112, 128, 144, and 160 h postinoculation. The use of a very low infective dose (2-20 oocysts) was sufficient to study the last merogony. The number of merozoites in meronts increased when the infective dose decreased. Only the first merogony of this coccidium in lymphocytes or M-cells of gut-associated lymphoid tissue (GALT) has previously been described. Three other generations of meronts are described herein. All these endogenous stages were observed in the epithelium of the vermiform appendix, sacculus rotundus, and Peyer's patches, especially at the bases of the domes. However, in heavily infected tissues the gamonts were seen throughout the epithelium of the GALT. The third- and fourth-generation meronts were of two types. As in other eimerian species of the rabbit, type A meronts produced thick polynucleated merozoites, whereas type B meronts gave rise to large numbers of thin merozoites with one nucleus. Microgamonts were polynucleated and less numerous than macrogamonts. Type A meronts were also polynucleated and less numerous at the end of the merogony. Therefore, types A and B could correspond to a sexual phenotype differentiation occuring during the two asexual phases of multiplication.     

Early pregnancy factor: its role in mammalian reproduction--research review

An immunosuppressive early pregnancy factor associated with mammalian reproduction is currently attracting considerable interest. Research into its detection, site of production, distribution, immunosuppressive property, characterization, and application is in progress in a number of laboratories. This review aims to crystallize the current research findings and to identify significant areas for further investigation and application.     

Re-entrant activity and its control in a model of mammalian ventricular tissue

We characterize the meander of re-entrant excitation in a model of a sheet of mammalian ventricular tissue, and its control by resonant drift under feedback driven stimulation. The Oxsoft equations for excitability in a guinea pig single ventricular cell were incorporated in a two dimensional reaction-diffusion system to model homogeneous, isotropic tissue with a plane wave conduction velocity of 0.35 m s-1. Re-entrant spiral wave solutions have a spatially extended transient motion (linear core) that settles down into rotation with an irregular period of 100-110 ms around an irregular, multi-lobed spiky core. In anisotropic tissue this would appear as a linear conduction block. The typical velocity of drift of the spiral wave induced by low amplitude resonant forcing is 0.4 cm s-1.     

Purification and characterization of ryanodine receptor 3 from mammalian tissue

The ryanodine receptors are intracellular Ca2+ release channels that play a key role in cell signaling via Ca2+. There are three isoforms. Isoform 1 from skeletal muscle and isoform 2 from heart have been characterized. Isoform 3 is widely distributed in many mammalian tissues although in minuscule amounts. Its low abundance has hampered its study. We now describe methodology to isolate mammalian isoform 3 in amounts sufficient for biochemical and biophysical characterization. Bovine diaphragm sarcoplasmic reticulum fractions enriched in terminal cisternae containing both isoforms 1 (>95%) and 3 (<5% of the ryanodine binding) served as starting source. Isoform 3 was selectively immunoprecipitated from the 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized fraction and eluted with peptide epitope. Isoform 3 thus prepared is highly purified as characterized by SDS-polyacryamide gel electrophoresis, Coomassie Blue staining, and by high affinity ryanodine binding. The purified isoform 3 was incorporated into planar lipid bilayers, and its channel properties were studied. Channel characteristics in common with the other two isoforms are slope conductance, higher selectivity to Ca2+ versus K+ (PCa/K approximately 6), and response to drugs and ligands. In its response to Ca2+ and ATP, it more closely resembles isoform 2. The first two-dimensional structure of isoform 3 was obtained by cryoelectron microscopy and image enhancement techniques.