A resonance frequency technique to determine elastic modulus of hydroxyapatite

A resonance frequency technique was applied to determine the elastic modulus of hydroxyapatite. A Free-Free Vibration Transducer was designed to determine elastic modulus in a longitudinal direction. A Fixed-Free Vibration Transducer was also designed to study elastic modulus of materials were specimens longer than 3 cm in length were not available. Six lots of hydroxyapatite were prepared utilizing the same process. The elastic modulus of hydroxyapatite varied between 3.94 x 10(10) (dyn/cm2) and 6.30 x 10(10) (dyn/cm2) in a longitudinal direction. For the same six lots, it varied between 1.95 x 10(10) (dyn/cm2) and 3.20 x 10(10) (dyn/cm2) in a cross direction. The elastic modulus values of cortical bone from dog tibias, fibulas, and femurs were also determined.     

The use of the preparation of F(ab')2 antibody from soluble immune complexes to determine the complexed antigens.

An autoradiographic technique for the characterization of antibody specificity in soluble antigen--antibody complexes has been developed. The circulating antigen--antibody complexes are precipitated by polyethylene glycol (PEG). The antibodies are liberated as F(ab')2 from the complexes by pepsin digestion. The antibody specificity against the putative antigen was revealed by radioimmunoelectrophoresis using [125I]-F(ab')2 reagents. The method was developed by using an experimental model of C3/anti-C3 complexes.     

Speckle correlation technique to determine roughness in the dermatologic interval

A non-invasive method is proposed to determine human skin roughness. The technique is based on measurement of the correlation between two field distributions scattered by a metallised triafol (cellulose acetate foil) replica of the epidermal area to be analysed. The two speckle patterns are produced from the same rough surface illuminated by two coherent plane waves (He-Ne laser) under two slightly different angles. The accuracy of the method is highlighted by measurements made on a set of standard samples with roughnesses previously determined by mechanical profilometry. Analysis of the results indicates a precision of around 10%, and an applicability within the interest range of very rough surfaces in excess of 4-5 microns.     

A new approach to determine the reliability of the rater technique by use of Smith's rating scales

Used as the base for the study a sample of ratings of 15 pilots made by supervisors during a 6-month period. This report of that evaluation confirms the validity of technique (Smith, 1974) using intraclass correlation and Spearman's rho to identify which raters were unreliable. More rigorous criteria for the elimination of unreliable raters were developed by applying the Kuder-Richardson reliability coefficient (KR-20) as a more precise measure of reliability than the intraclass correlation criteria used by Smith. The proposed technique permits improved reliability by the criterious removal of raters who are not contributing significantly to the total consistency of the behavioral scale and thus eliminates partially the inconvenience of the unavailable significant table of data to test the acceptance of the reliability.     

A technique to tocopherol (vitamin E) histochemical detection

A technique to detect tocopherol histochemically was proposed in basis on the following schedule: 1. toluidine blue and Schiff reagent negativity before a suitable oxidation; 2. performic acid-toluidine blue and performic acid-Schiff reagent positivity after fixing in formalin-CaCl2; 3. performic acid-toluidine blue and performic acid-Schiff reagent negativity after fixing in formalin-HgCl2; 4. ferric ferricyanide reaction positivity not influenced by the formalin-HgCl2 blockade. This technique tested on filter paper strips loaded with several lipids, steroids, vitamins, proteins, amino acids and ribonucleic acid shows that it is specific to tocopherols among the tested substances. Used on tissue sections this technique appears as very suitable to histochemical purposes.     

Circulating immune complexes, detection in human glomerulonephritis by the PEG-EDTA technique (author's transl)

In a prospective study, we screened sera samples of 128 patients with glomerulonephritis (GN) for the presence of circulating immune-complexes (CIC) fixing C1q by precipitation of native C1q with 2% polyethylene glycol (PEG) in the presence of 10 mM EDTA. The amount of precipitated C1q, as measured by Mancini, is less than 27% (m +/- 2 SD) of the original value in control sera. We found 35/128 (27%) positives samples, distributed as follows: 4/17 (24%) in acute GN, 10/15 (67%) in SLE, 2/16 (13%) in membraneous GN, 13/24 (54%) in membranoproliferative GN, 3/16 (19%) in segmental focal hyalinosis, 2/10 (20%) in minimal lesions nephrotic syndrome, and 1/30 (3%) in mesangial IgA GN. We then correlated these results with clinical, serological and pathological data. Whatever the type of GN, the presence of CIC fixing C1q correlated significantly, well with : the presence of chronic renal failure (serum creatinine greater than or equal to 2 mg/dl) (X2 = 5.48, p less than 0.02), the presence of hypocomplementemia (X2 = 12.30, p less than 0.001), the presence of low serum C3 (X2 = 8.25, p less than 0.01), the activation of C3 through normal pathway (low serum C4 and/or C1q) (X2 - 18.12, p less than 0.001) and the presence of glomerular deposits of C3 (X2 = 8.52, p less than 0.01), of C4 (X2 = 7.10, p less than 0.01), and of C1q (X2 = 4.11, p less than 0.05). The technique is simple, does not require labeled C1q, and allows the further study of antigen or antibody determinants of the complexes. Its sensitivity is near that of the 125 I-C1q binding assay technique. Such routine screening is a major immunopathologic step in the investigation of human GN.     

Comparison of the FLOTAC technique with the McMaster method and the Baermann technique to determine counts of Dictyocaulus eckerti L1 and strongylid eggs in faeces of red deer (Cervus elaphus)

The FLOTAC flotation technique has been introduced as a new diagnostic tool to detect parasitic elements from faeces. Samples from naturally infected young deer were used for counting Dictyocaulus larvae and strongylid eggs. The FLOTAC technique, using 11 different flotation solutions with specific gravities (sg) between 1.20 and 1.45, was compared with the Baermann technique and the saturated sodium chloride (sg 1.20)-based McMaster method. In addition, a comparison was made between the FLOTAC technique with magnesium sulphate (sg 1.28) and the Baermann technique for larval recovery from faeces that were examined on the day of collection or after 7 days storage at 4°C. On the whole egg counts between the FLOTAC using different flotation solutions and the McMaster were unremarkable. In contrast, variations of larval counts were detected between different flotation solutions as well as with the Baermann technique. Most flotation solutions with a specific gravity of 1.20 floated significantly fewer lungworm larvae (p < 0.05) compared to flotation solutions with a higher specific gravity. Magnesium sulphate (sg 1.28) consistently produced the highest mean larval counts in all conducted experiments. Larval counts using magnesium sulphate (sg 1.28) were higher than with the Baermann technique both on the day of collection and after 7 days. Overall, the use of magnesium sulphate (sg 1.28) with FLOTAC for larval counts resulted in higher counts than the Baermann recovery technique and was the better choice of those flotation solutions examined. Furthermore, magnesium sulphate (sg 1.28) was also reliable for strongylid egg detection with the FLOTAC apparatus.     

A continuous-flow technique for analysis of stoichiometry and transport kinetics of gastric H,K-ATPase

A continuous-flow method was developed for determining the stoichiometry of the gastric proton pump H, K-ATPase (EC 3.6.1.36) in its hydrolysis of ATP and translocation of H⁺ and the K⁺ congener “Rb⁺. H, K-ATPase-containing vesicles which had been isolated from pig gastric mucosa were incubated at 37 °C for 2 h in 150 mM ⁸⁶RbCl, 0.5 mM ethylenebis(oxyethylenenitrilo)tetra-acetic acid and J mM 2–(N-morpholino)ethane sulphonic acid (Mes) adjusted to pH 6.1 with Tris, and then applied onto a 0.45 μm pore size cellulose acetate filter. The immobilized vesicles were perfused with 0.15 mM Mes/Tris buffer, pH 6.1, containing 150 mcholine chloride and 0.2 mM MgCl₂., After changing to a medium containing 0.1 mM ATP, the amounts and rates of H⁺ uptake, ⁸⁶Rb⁺ efflux and ATP hydrolysis were measured. The initial ratio of Rb⁺ transported to ATP hydrolysed gave values of 0.96 ± 0.26 (mean f SD, n= 28). The initial ratio of ATP-dependent Rb⁺ efflux to H⁺ uptake gave values of 0.92 + 0.28 (mean f SD, n= 28). The Mg-ATPase activity was measured in vesicles which had been incubated with choline chloride instead of RbC1. This activity was 15.8 f 8.7% (mean + SD) of the total ATPase activity in the initial fractions used for calculation of the stoichiometry. It is argued that this Mg-ATPase may be an intrinsic activity of the H, K-ATPase and that the relation between these activities is dependent on the amount of K⁺ (or Rb⁺) present in the assay. However, whether corrections were made for this Mg-ATPase or not, it had only marginal effects on the calculations of the stoichiometry of the pump. Thus simultaneous measurements of ⁸⁶Rb⁺ efflux, H⁺ uptake and ATP hydrolysis in immobilized gastric vesicles gave a stoichiometry of the pump close to a 1:1:1 ratio. These results indicate that the pump is non-electrogenic.     

Adaptation to the Biomek 1000 workstation (Beckman) of the MHA-TP technique (Ames)

The microhemagglutination-Treponema pallidum (MHA-TP) was automated with the Biomek 1000 workstation (Beckman). 3,000 serum specimens were studied with manual and automated procedures. Thirty nine serum specimens were found positive by both techniques, neither false positive nor false negative results were observed with the Biomek 1000 workstation. The statistical analyse showed no differences between the two procedures, variations were observed only with low titer sera. In conclusion, time saving and augmentation of productivity obtained by automation of this method could be interesting for a microbiology laboratory.     

A continuous-flow technique for analysis of stoichiometry and transport kinetics of gastric H,K-ATPase

A continuous-flow method was developed for determining the stoichiometry of the gastric proton pump H,K-ATPase (EC 3.6.1.36) in its hydrolysis of ATP and translocation of H+ and the K+ congener 86Rb+. H,K-ATPase-containing vesicles which had been isolated from pig gastric mucosa were incubated at 37 degrees C for 2 h in 150 mM 86RbCl, 0.5 mM ethylenebis(oxyethylenenitrilo)tetra-acetic acid and 3 mM 2-(N-morpholino)ethane sulphonic acid (Mes) adjusted to pH 6.1 with Tris, and then applied onto a 0.45 micron pore size cellulose acetate filter. The immobilized vesicles were superfused with 0.15 mM Mes/Tris buffer, pH 6.1, containing 150 mM choline chloride and 0.2 mM MgCl2. After changing to a medium containing 0.1 mM ATP, the amounts and rates of H+ uptake, 86Rb+ efflux and ATP hydrolysis were measured. The initial ratio of Rb+ transported to ATP hydrolysed gave values of 0.96 +/- 0.26 (mean +/- SD, n = 28). The initial ratio of ATP-dependent Rb+ efflux to H+ uptake gave values of 0.92 +/- 0.28 (mean +/- SD, n = 28). The Mg-ATPase activity was measured in vesicles which had been incubated with choline chloride instead of RbCl. This activity was 15.8 +/- 8.7% (mean +/- SD) of the total ATPase activity in the initial fractions used for calculation of the stoichiometry. It is argued that this Mg-ATPase may be an intrinsic activity of the H,K-ATPase and that the relation between these activities is dependent on the amount of K+ (or Rb+) present in the assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibody-based ELISA to quantify the major allergen of Cynodon dactylon (Bermuda grass) pollen, Cyn d 1

BACKGROUND: Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major allergen Cyn d 1, a glycoprotein composed of a number of isoforms with a molecular mass of 31-32 kDa. The aim of this work was to develop a monoclonal antibody (mAb)-based ELISA to quantify Cyn d 1, and to assess the correlation of the allergen content with the biological activity of C. dactylon pollen extracts. METHODS: After fusion of myeloma cells with spleen cells from a BALB/c mouse immunized with C. dactylon pollen extract, Cyn d 1-specific mAbs secreting hybridomas were selected, and the antibodies characterized. One of them (4.4.1) was used as the capture antibody in an ELISA method for Cyn d 1 quantitation. An anti-Cyn d 1 rabbit serum was used as the second antibody. Cyn d 1 was purified by immunoaffinity chromatography with mAb 4.4.1, characterized, and used as the standard in the assay. RESULTS: The identity, purity and isoallergen composition of affinity-purified Cyn d 1 was confirmed by N-terminal amino acid sequencing, SDS-PAGE, Western blot and 2D electrophoresis. The Cyn d 1 ELISA is highly specific and sensitive, with a detection limit of 0.24 ng/ml and a linear range of 1.1-9.2 ng/ml. An excellent correlation was found when the content of Cyn d 1, measured in 16 different extracts, was compared with the allergenic activity of the same extracts determined by RAST inhibition. CONCLUSIONS: The results prove the usefulness of the Cyn d 1 ELISA for the standardization of C. dactylon-allergen products on the basis of major allergen content.     

Gel electrophoresis of native gelsolin and gelsolin-actin complexes

Summary BHK gelsolin migrated on non-denaturing 8-25% polyacrylamide gels with an apparent molecular mass of 80 kDa. In the absence of Ca²⁺ no complex formation occurred between BHK gelsolin and actin. In the presence of Ca²⁺ two complex species were found: a ternary complex, GA₂, of apparent molecular mass of 210 kDa at gelsolin: actin ratio of 12, and a novel quaternary complex, GA₃, of apparent molecular mass of 247 kDa when actin was in excess. Both cytoplasmic and plasma gelsolin species form GA₃ with skeletal muscle actin. No complexes larger than GA₃ were observed. The formation of GA₃ involves the binding of a third actin to the gelsolin molecule at the site previously assumed to be masked, rather than to the actin molecules already present in GA₂. In preference to GA₃, GA₂ was incorporated into actin filaments stabilized with phalloidin. On chelation of free Ca²⁺, both GA₂ and GA₃ dissociated to form the EGTA stable binary complex (GA) with an apparent molecular mass of 140 kDa and free actin.     

XRCC1 coordinates disparate responses and multiprotein repair complexes depending on the nature and context of the DNA damage

XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both short patch (SP) and long patch (LP) base excision repair (BER). We show that POLβ and PNK colocalize with XRCC1 in replication foci and that POLβ and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied. We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLβ and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLβ to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER proteins to sites of DNA damage and provide evidence for a role of XRCC1 in the organization of BER into multiprotein complexes of different sizes.     

A study to determine the incidence of estrogen receptor (ER) and progesterone receptor (PR) expression in different histological grades of breast cancer

Abstract

Carcinoma of the breast is the most common non-skin malignancy in women and second most common cause of death due to cancer in females. Breast cancer is a heterogeneous disease with varied morphological appearances, molecular features, behavior, and response to therapy. This study was conducted with the aim of analysis of steroid receptor status in breast cancer and its association with other prognostic factors of breast cancer such as histological grade, tumor size, and axillary lymph node status. Fifty patients with the diagnosis of breast cancer were included in this study. Detailed clinical and histopathologic data were recorded in all cases. Estrogen receptor (ER) and progesterone receptor (PR) status was evaluated by immunohistochemistry. Histological grading was done with Nottingham modification of the Bloom and Richardson method. Fisher’s exact test and chi-square test was used for statistical analysis. On immunohistochemical staining, 44% cases proved to be ER positive and 48% cases PR positive. The results from the present study in India documented low ER and PR positivity in breast cancer in comparison to results from the western world. An inverse relation was found between hormone receptor (HR) status and histological grading, but no statistically significant relationship was noted between HR expression and tumor size and lymph node status.     

Binding of SLE autoantibodies to native poly(I), ROS-poly(I) and native DNA: a comparative study.

Reactive oxygen species (ROS) are implicated in a variety of human diseases. The formation of pathogenic anti-DNA antibodies in systemic lupus erythematosus (SLE) has been extensively investigated. ROS-modified DNA has been found to be a better antigen for anti-DNA antibodies found in SLE sera. A comparative binding of SLE autoantibodies with native poly(I), ROS-poly(I) and nDNA has been studied. Affinity-purified SLE IgG exhibited a high degree of specificity towards the ROS-modified poly(I) in comparison to native DNA and native poly(I), reiterated visually by gel retardation assay. The data suggested that hydroxyl radical-modified nucleic acids like RNA and DNA might be agent for the induction of circulating SLE anti-DNA autoantibodies.     

A technique for the purification of immune complexes using rheumatoid factor.

A method for the purification of immune complexes (IC) from human serum is described, using as a model complexes of tetanus toxoid with human antitoxoid antibodies (TAT). The technique is based on the ability of IC to bind to tubes coated with rheumatoid factor (RF). Small amounts of TAT IC were added to human serum and the mixtures were rotated overnight in tubes coated with RF. The tubes were then washed and the bound material was eluted with sodium dodecyl sulphate and iodinated with 125I. Analysis of the labeled preparation by electrophoresis in polyacrylamide gels revealed the presence of the toxoid component. The technique should be useful in isolating small amounts of IC for analytical purposes.     

A novel technique to collect root exudates from mustard (Brassica juncea)

A very simple, novel, cost effective, easy to use technique has been developed for the collection of root exudates from small seeded plants, under laboratory conditions. 200–1000 μl micro tips (Tarsons), kept in 100 ml glass beakers, were used as holders for the small seeds of Indian mustard (Brassica juncea) and the exudates were trapped in liquid culture medium. The exudates, so obtained, were authenticated and analyzed for organic compounds such as sugars, amino acids and organic acids, as well as chemotactic response towards rhizobacteria. Method was found to be suitable and easy to handle for small seeds. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Epitope-tagging approach to determine the stoichiometry of the structural and nonstructural proteins in the virus particles: amount of Vpr in relation to Gag in HIV-1

We used an epitope-tagging approach to determine the ratio of Gag (structural) to Vpr (nonstructural) in the virus particles directed by human immunodeficiency virus type 1. For this purpose, chimeric Gag and Vpr expression plasmids were constructed with the Flag epitope (DYKDDDDK), and the sequences corresponding to the chimeric protein were introduced into human immunodeficiency virus type 1 proviral DNA (NL4-3) to determine the ratio in the virus particles when these proteins are expressed in cis. In addition, NL4-3 DNA was modified to disrupt Vpr synthesis to determine the extent of incorporation of Vpr-FL when it is expressed in trans through a heterologous promoter. The analysis of virus particles generated by transfection of proviral DNA into RD cells indicated that (1) the ratio of Gag to Vpr in virus particles, when Vpr-FL is expressed in cis (in the context of proviral DNA), is in the range of 150-200:1 (14-18 molecules of Vpr per virion) and (2) the expression of Vpr-FL in trans showed efficient incorporation with a Gag to Vpr ratio of 5-7:1 (392-550 molecules of Vpr). These results suggest that the presence of the same epitope on different viral proteins may provide an accurate comparison of these proteins in the virus particles.