Kinetic differences in the phospholamban-regulated calcium pump when studied in crude and purified cardiac sarcoplasmic reticulum vesicles

Phospholamban (PLN) phosphorylation contributes largely to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. The mechanical effects of PLN phosphorylation on the heart are generally attributed solely to an increase in the apparent affinity of the Ca pump in the sarcoplasmic reticulum (SR) membranes for Ca2+ with little or no effect on Vmax(Ca). In the present report, we compare the kinetic properties of the cardiac SR Ca pump in commonly studied crude microsomes with those of our recently developed preparation of light SR vesicles. We demonstrate that in crude microsomes, the increase in the apparent affinity of the pump for Ca2+ is larger, while the increase in Vmax(Ca) is smaller, than in purified vesicles. The greater phosphorylation-induced increase in apparent Ca2+ affinity in crude microsomes may be further enhanced by an ATP-sensitive inhibitory effect of ruthenium red on the activity of the pump at subsaturating, but not saturating, Ca2+ concentrations as a result of a greater inhibition in unphosphorylated microsomes. Upon increasing the ATP concentration from 1 to 5 mm, an inhibition by 10 micrometer ruthenium red is eliminated in phosphorylated microsomes and reduced in control microsomes. Addition of the phosphoprotein phosphatase inhibitor okadaic acid produces a considerable increase in the phosphorylation-induced effects in both crude and purified microsomes. We conclude that the use of purified cardiac SR vesicles is critical for the demonstration of a major increase in Vmax(Ca) in addition to an increase in the pump's apparent affinity for Ca2+ in response to phosphorylation of PLN by protein kinase A.     

Immuno-isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting

Immuno-isolation is a powerful technique for the isolation of cells as well as subcellular organelle populations based on their antigenic properties. We have established a method for immuno-isolation of peroxisomes (PO) from both rat liver and the human hepatoblastoma cell line HepG2 using magnetic beads as solid support. A polyclonal antibody raised against the cytoplasmic C-terminal 10 amino acids of the rat 70 kDa peroxisomal membrane protein was covalently bound to magnetic beads (Dynabeads M-450). The coated beads were incubated with a light mitochondrial fraction and the organelle-bead complexes formed were separated by magnetic sorting in a free-flow system without pelleting the complexes during the isolation procedure. Scanning electron microscopy revealed decoration of beads with particles measuring 150-400 nm in diameter. The particles were identified as PO by catalase cytochemistry and biochemically by marker enzyme analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as immunoblotting for specific detection of peroxisomal matrix, core and membrane proteins. The functional significance of PO in man is emphasized by the existence of inherited diseases such as the Zellweger syndrome in which intact PO are lacking, but peroxisomal remnants called "ghosts" are observed instead. Peroxisomal disorders are usually studied using skin fibroblast cell lines derived from afflicted patients and immuno-magnetic separation may prove particularly useful for the investigation of such cultured cells and for further elucidation of the pathogenesis of fatal peroxisomal disorders.     

Studies on Alternaria allergens. II. Measurement of the relative potency of commercial Alternaria extracts by the direct RAST and by RAST inhibition

We investigated 104 cases of infantile convulsions which occurred before two years of age. These cases had no detectable signs of acute brain disorders, and they had normal psychomotor development and normal EEG findings at onset. (1) Sex ratio was 1:1. (2) A positive family history of various types of convulsions was obtained in 30.8% (Febrile convulsion: 65.7%) (3) The incidence of perinatal anoxic brain damage was higher than that in the control group. This was probably related to the incidence of infantile convulsion. (4) More than half the cases (66.3%) had the first convulsion between three and eight months of age, and 95.6% of prospectively follow-up cases were shown free from attacks before two years of age. (5) Precipitating factors to convulsion were found in 45.1% of all cases. These factors were bathing and non-febrile infection. (6) The most common form of infantile convulsion is generalized tonic and/or clonic convulsion (78.8%) within 5 min. (82.7%). This convulsion reccurs frequently in a relatively short period. (7) The incidence of subsequent epilepsy seems to have been found among patients who had had an initial attack between three and five months of age, and had then experienced convulsion more than six times or in a prolonged state.     

Can active immunization redirect an anti-IgE immune response?

We used as a template a mouse monoclonal antibody against IgE to isolate peptides from random peptide phage display libraries. Thereby, two types of peptides were isolated that corresponded to two different epitopes on the human IgE molecule. These peptides, also called mimotopes, seem to be a suitable tool in conjunction with carriers to induce an autoimmune response with a beneficial effect in humans, because the originally used template antibody is capable of neutralizing IgE, is nonanaphylactogenic, and inhibits IgE synthesis. The vaccination approach is further supported by the fact that we were capable of isolating anti-idiotypic antibodies from antibody phage display libraries against the template antibody. These anti-idiotypic antibodies were inhibited by both of the isolated IgE mimotopes. Thus, active vaccination with defined IgE mimotopes may represent a follow-up drug for the presently used anti-IgE antibodies.     

Characterization of complex formation by humanized anti-IgE monoclonal antibody and monoclonal human IgE

The interaction of human IgE with high-affinity IgE Fc receptors on cells of the immune system plays an essential role in the type I hypersensitivity reaction. A proposed therapy is to use an anti-IgE monoclonal antibody to block the binding of IgE to its high-affinity receptor on mast cells and basophils, thus preventing subsequent release of the inflammatory agents after exposure to allergen. We report here the solution characteristics of immune complexes formed by a humanized anti-IgE monoclonal antibody (rhuMAb E25) and IgE using sedimentation analysis and size exclusion chromatography. We demonstrate that the rhuMAb E25 is able to form a variety of complexes with IgE at different molar ratios. The largest complex was identified by sedimentation equilibrium analysis as a heterohexamer with very high stability. The intermediate complex formed when one of the interacting components is in large molar excess appears to have a trimeric structure. The high-affinity interaction of rhuMAb E25 and IgE has also been confirmed. Furthermore, by using hydrodynamic modeling, we show that the largest complex may be represented by a cyclic structure.     

Optimal data selection in the reduced array selection task (RAST).

The predictions of M. Oaksford and N. Chater's (see record 1995-08271-001) optimal data selection (ODS) model for the reduced array selection task (RAST) were tested in 4 experiments. Participants tested a hypothesis, if p then q, by selecting cards showing q or not q instances. In Experiment 1, where selections were made from different sized stacks of q and not q cards, as P(q) increased, not q card selections rose, and q card selections fell, as predicted. Experiment 2 controlled for the possibility that stack height influenced responses; these results were also consistent with ODS. Experiment 3, which controlled further for this possibility, replicated Experiment 1. Experiment 4 addressed a final issue concerning the medium P(q) condition by concentrating on initial card selections; the results were again consistent with ODS. Although generally consistent with the ODS model, these experiments also suggest some interesting revisions. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Inhibitory effects of an anti-IgE antibody E25 on allergen-induced early asthmatic response

Inhaled allergens, acting through IgE-dependent mechanisms, are important triggers of asthma symptoms and inducers of airway hyperresponsiveness and airway inflammation. The effect of anti-IgE recombinant humanized monoclonal antibody-E25 (rhuMAb-E25) on the provocation concentration of allergen causing a 15% fall in FEV1 (allergen PC15) during the allergen-induced early asthmatic response (EAR) was assessed in a multicenter, randomized, double-blind, parallel group study. Ten of 11 allergic asthmatic subjects randomized to receive intravenous rhuMAb-E25, 2 mg/kg on study day 0 and 1 mg/kg on Days 7, 14, 28, 42, 56, and 70 completed the study; nine received intravenous placebo. The allergen PC15 was measured on Days -1, 27, 55, and 77 and methacholine PC20 on Days -2, 42, and 76. rhuMAb-25 was well tolerated and only one patient (active group) was withdrawn because of a generalized urticarial rash after the first dose. Compared with baseline values (Day -1), the median allergen PC15 on Days 27, 55, and 77 were increased by 2.3, 2.2, and 2.7 doubling doses (delta log PC15/0.3) respectively with rhuMAb-E25 and -0.3, +0.1, and -0.8 doubling doses with placebo (p < or = 0.002). Methacholine PC20 improved slightly after rhuMAb-E25, this change becoming statistically significant on Day 76 (p < 0.05); no change was observed in the placebo group. Mean serum-free IgE fell by 89% after rhuMAb-E25 while there was no significant change after placebo. The inhibitory effects of rhuMAb-E25 on allergen-induced EAR suggest that it may be an effective, novel antiallergic treatment for asthma.     

Extracts from various mite species contain cross-reactive and noncross-reactive IgE epitopes. A RAST inhibition study

PURPOSE: A low pregnancy rate in in vitro fertilization (IVF) patients of advanced maternal age may be caused by aneuploidies originating from non disjunction in the first or second meiotic divisions. We introduced genetic testing of oocytes by sampling and fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies, to avoid fertilization and transfer of aneuploid oocytes in IVF patients of advanced maternal age. METHODS: Three hundred and sixty-three IVF patients 34 years and older participated in the study. Using micromanipulation procedures, the first and second polar bodies were removed following their extrusion from the oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21 to detect oocytes with common aneuploidies. RESULTS: Of a total of 538 IVF cycles, 3250 oocytes were available for FISH analysis, with conclusive FISH results in 2742 oocytes (84.3%). As many as 1102 (40%) of oocytes were predicted to be aneuploid and not transferred. Of 1640 embryos predicted to be normal, 1145 were transferred in 467 treatment cycles, resulting in 107 pregnancies (23%), from which 67 healthy children have been born, 32 pregnancies spontaneously aborted, and 15 pregnancies are ongoing after being confirmed normal by prenatal diagnosis. CONCLUSIONS: Preimplantation diagnosis by first- and second-polar body FISH analysis allows us to avoid the age-related risk of common aneuploidies in IVF patients of advanced maternal age.     

Determination of specific IgE and IgG serum antibodies during immunotherapy in hay fever patients by RAST

A solid phase radio-immunoassay, based on the commercially available antigen preparations on paper discs supplied with the radio-allergosorbent test (Phadebas RAST, Pharmacia) for semi-quantitative determination of specific IgE has been applied to the demonstration of allergen-specific antibodies of the IgG class in sera of hay fever patients. A significant rise of specific IgG antibody has been observed regularly as a result of successful immunotherapy. The rise of such "blocking antibodies" occurred within 3 months after commencement of immunotherapy (IT), well before nasal provocation tests became negative in the patients. No significant decrease of specific IgE antibodies was observed over the one year observation period. While these data confirm earlier results by other groups concerning the appearance of blocking antibodies of IgG class as a result of IT, thus providing additional evidence for its mechanism, they also offer a convenient serological method for monitoring the results of IT.     

The nature and use of purified insulins

The limited availability of highly purified insulin preparations for the treatment of diabetes mellitus in australia since 1975, has prompted a review of their history, clinical use and potential in the treatment of diabetics receiving insulin.     

Effect of crude fat and crude protein on reproduction and weaning growth in four strains of inbred mice

Diets made from natural ingredients were fed to four inbred strains of mice (BALB/cAnN, C3H/HeN, C57BL/6N and DBA/2N) to study the effects of different concentrations of dietary crude protein, 18% and 24% with crude fat concentrations of 4%, 8%, and 12% on reproduction and weanling growth. The parameters measured included the number of litters and pups born, the number of litters and pups weaned, weanling mortality and weanling weight. Neither crude protein nor crude fat concentrations had significant effects on any of the reproductive parameters tested. However, a significant fat x protein interaction was observed for reproduction. These results indicate that the absolute concentrations of crude protein and crude fat in diets for inbred mouse production are not as important as the ratio of these two nutrients. There was also a differential response in reproduction among the strains due to the level of fat which indicates different dietary requirements for fat among these four inbred strains for maximum reproduction. In contrast to the results for reproduction, crude protein and crude fat independently had significant effects on weanling weight, but there was no effect due to the ratio of the nutrients. There was a significant increase in the weanling growth rate for all four strains as the dietary fat level increased, but a decrease in growth rate as the protein level increased. The reduced growth rate due to the increased protein was not of the same magnitude for all four strains which indicates specific protein requirements among the strains for weanling growth.     

Analysis of anti-IgE and allergen induced human basophil activation by flow cytometry. Comparison with histamine release

OBJECTIVE AND DESIGN: On the basis of flow cytometric methods previously described for the analysis of human basophil activation, we present here a bi-color anti-IgE FITC, anti CD63 PE method and the correlation with histamine release. MATERIALS AND SUBJECTS: Subjects allergic to grass pollen were selected by their clinical history, skin tests and specific IgE. METHODS: Basophils gated in the lymphocyte region of the side scatter (SSC)/forward scatter (FSC) pattern were selected by their high IgE epitope density. Percentage of cells expressing CD63 marker, upregulated on activated basophil membrane, was calculated by the cytometer. Histamine released into the supernatants was measured by RIA. RESULTS: In these conditions, flow cytometric analysis of blood leukocytes showed that the selected cells had the phenotype CD14-, CD19-, CD45+, IgE++ and CD63- or + which is related to human basophil phenotype, the isotype controls being negative. The use of an anti-CD41 FITC antibody also showed the presence of aggregated platelets on the basophil membrane, CD63 antigen being, however, expressed by basophils themselves and not by platelets. Moreover, no statistical difference was observed between histamine release and flow cytometry after passive sensitization of blood donor leukocytes. CONCLUSION: Flow cytometry, as a popular method often used in the immunology and haematology departments of clinical laboratories may represent a new alternative for allergy diagnosis and basophil pharmacology.     

Diagnostic use of enzymatic RAST skin tests and determination of eosinophils in nasal mucosa in allergic rhinitis

AIMS: Allergic rhinitis is the most frequent disease mediated by immunoglobulin E (IgE). Nasal challenge is the gold standard for the diagnosis of allergic rhinitis. Skin tests (ST) are the most used diagnostic method to detect the presence of specific IgE bind to skin mast cells. The exposition of the nasal mucous membrane to the allergen is followed by an increase of the local eosinophils; the count of eosinophils in nasal mucous (ENM) is a diagnostic test for allergic rhinitis. Enzymatic RAST or enzymatic allergo-sorbent test (ESA) measures the level of serum allergen-specific IgE. OBJECTIVE: To measure the sensitivity, specificity, and diagnostic precision of ST, EAST and ENM in allergic rhinitis. METHOD: We studied 241 individuals, 162 of them had allergic rhinitis, and 79 were healthy controls. They underwent nasal challenge and intradermic ST for Dermatophagoies spp (acarus). Fraxinus americana (Ash-tree), Amaranthus palmieri (quelite), Cynodon Dactylon (capriola) and Felis catus (cat), EAST for Dermatophagoides pteronyssinus (acarus), and ENIVI. Results of ST, EAST and ENIVI were compared with their corresponding nasal challenge, and the prevalence of allergic rhinitis for each allergen was calculated. The best cut point was assessed by means of receiver-operator curves (ROC), and sensitivity, specificity, positive predictive value, negative predictive value, inter-observer concordance coefficient, area under ROC (0), standard error of 0 (SEO), and 95% confidence interval of 0 of each test were calculated using the best cut point. RESULTS: ST and EAST had the best sensitivity and specificity. ENM had the lowest sensitivity and specificity. CONCLUSION: For the diagnosis of Dermatophagoides spp allergic rhinitis ST for Dermatophagoides spp and EAST for Dermatophagoides pteronyssinus have the same diagnostic precision. According to the indexes for diagnostic precision, and inter-observer concordance coefficient, ST and EAST are useful to diagnose allergic rhinitis induced by the evaluated allergens. ENIVI is a test that is not very useful for the diagnosis of allergic rhinitis.     

Flow cytometric assay for cytotoxic activity of crude Clostridium perfringens enterotoxin using non-adherent cell FM3A

The flow cytometric assay method was tested for the cytotoxic activity of Clostridium perfringens enterotoxin (CPE) in culture using mouse mammary carcinoma cell line FM3A stained with propidium iodide (PI). From the results obtained, FM3A cells proved to be susceptible to CPE. A reproducible dose-response curve with FM3A was obtained between crude CPE at 13.9-109 ng/ml and between purified CPE at 40-400 ng/ml, respectively. These findings indicate that non-adherent FM3A is preferable to determine the cytotoxic activity of CPE because it can be used without detachment procedures with trypsinin compared with adherent African monkey kidney cell line (Vero cells). Furthermore, the flow cytometry with non-adherent cell FM3A stained with PI only proved to be a useful method to determine the biological activity of CPE in culture isolates.     

采用转炉吹炼粗银粉工艺提高银产品质量

铜阳极泥经亚钠分银生产的粗银粉中Bi、Te、Pb、Sb等杂质含量较高,用湿法工艺难将这些杂质与主金属分离,影响电银产品质量,粗银粉采用转炉吹炼-电解生产工艺,除杂效果理想,电银产品质量稳定提高,获得了较好的技术经济指标。     

Contents of lecithin and choline in crude drugs

The determination of lecithin and choline in crude drugs was established by a combination of high performance liquid chromatography (HPLC) with electrochemical detector (ECD) and enzyme reaction. Lecithin in crude drugs extracted with a mixture of chloroform-methanol (2:1) at room temperature was hydrolyzed by phospholipase D. The hydrolyzate was injected to HPLC, and choline was separated from impurities by reverse phase column. The choline was converted to betaine and hydrogen peroxide by passing through column packed with immobilized choline oxidase. This hydrogen peroxide was detected by ECD. The peak area of hydrogen peroxide derived from lecithin was proportional to the concentration of lecithin from 0.10 to 1.52 microgram/ml. Choline in crude drugs was extracted with ethanol under reflux and determined under the same HPLC conditions as lecithin. The peak area of hydrogen peroxide derived from choline was proportional to the concentration of choline from 0.01 to 0.45 microgram/ml. The contents of lecithin and choline in 31 kinds of crude drugs were determined by these established methods. The results showed that Cervi Parvum Cornu, Kokurozin, Foenigraeci Semen and Psoraleae Semen contained more lecithin than other crude drugs, while Angelicae Radix, Foenigraeci Semen, Psoraleae Semen, and especially Hippocampus were found to contain more choline than other crude drugs.     

Multiple IgE-mediated sensitizations to enzymes after occupational exposure: evaluation by skin prick test, RAST, and immunoblot

We investigated 10 sensitized and 10 nonsensitized workers from a pharmaceutic factory who had been exposed to powdered trypsin, chymotrypsin, bromelain, papain, amylase, and lipase. Ten nonallergic subjects served as a control group. Titrated skin prick tests (SPT), RAST, and immunoblot studies were performed with all six enzymes. SPT reactivity revealed multiple sensitizations to proteolytic enzymes, i.e., papain (specifically sensitized/total number of sensitizations: 9/10), trypsin (8/10), chymotrypsin (8/10), and bromelain (7/10) and appeared to be more frequent and more pronounced than sensitizations to amylase (3/10) or lipase (3/10). The low molecular weight of proteolytic enzymes (20-30 kDa) and their biologic activity might facilitate mucosal penetration more easily and thus-compared to amylase and lipase-permit an immune response and induction of allergic hypersensitivity. Immunoblot studies demonstrated IgG-binding bands in both SPT-positive and -negative workers, indicating exposure to the enzymes, but not in 10 unexposed control subjects. IgE-binding bands of the enzymes were detected only in workers with a positive SPT reaction and/or a positive RAST result. IgG bands were more frequent and the IgG/IgE ratio was increased in workers without allergic complaints compared to symptomatic workers. This might indicate that high levels of specific IgG antibodies to enzymes are associated with an immune response lacking allergic manifestations in spite of IgE-mediated sensitizations to the enzymes. Atopic subjects were at greater risk of developing IgE-mediated sensitization (7/10) and allergic symptoms to enzymes (5/7). However, even without risk of atopy, IgE-mediated hypersensitivity occurred in a few subjects (3/13) exposed to enzymes by inhalation for prolonged periods of time.