Membrane Strip Research Articles

Plastic ELISA-on-a-Chip Based on Sequential Cross-Flow Chromatography

A plastic chip that can perform immunoassays using an enzyme as signal generator, i.e., ELISA-on-a-chip, was developed by incorporating an immunostrip into channels etched on the surfaces of the chip. To utilize an analytical concept of cross-flow chromatography, the chip consisted of two cross-flow channels in the horizontal and vertical directions. In the vertical channel, we placed a 2-mm-wide immunostrip for cardiac troponin I (cTnI), which was identical to a conventional rapid test kit except for the utilization of an enzyme, horseradish peroxidase (HRP), as tracer. An enzyme substrate supply channel and a horizontal flow absorption pad compartment were transversely arranged on each lateral side of the signal generation pad of the strip, respectively. Upon application of a sample containing cTnI, it migrated vertically through the membrane strip by capillary action, and antigen-antibody binding occurred. After 15 min, the horizontal flow was initiated by the addition of a chromogenic substrate solution for HRP into the supply channel and by partial superimposition of the horizontal flow absorption pad onto the signal generation pad. A color signal proportional to the analyte concentration was produced on this pad, measured after 5 min as optical densities using a digital camera-based detector, and quantified by integration of the densities under the peak after normalization. Its calibration curve indicated that the detection limit of the chip was approximately 0.1 ng/mL and its quantification limit was 0.25 ng/mL. In measuring blindly prepared samples, the chip performance correlated with that of a reference system, Beckman Coulter Access, within 2.5-fold discrepancy at the detection limit.

Decreased Subunit Stability as a Novel Mechanism for Potassium Current Impairment by a KCNQ2 C Terminus Mutation Causing Benign Familial Neonatal Convulsions

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (I(KM)), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043deltaT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043deltaT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 microm) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043deltaT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates.

Colloidal gold-based immumochromatographic assay for detection of diethylstilbestrol residues

One-step membrane-based competitive colloidal gold-based immunoassays in immunochromatographic formats for the rapid detection of diethylstilbestrol (DES) were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and DES hapten-ovalubumin conjugate (test line). Anti-DES polyclonal antibody labeled with colloidal gold particles was first incubated with DES. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for immunochromatographic of 0.5 microg/kg for detecting DES standard solution, and the limit of detection was 5 microg/kg for detecting the DES spiked in swine pork and liver. The assay time for test was less than 5 min, suitable for rapid testing on-site.

Development of a rapid, immunochromatographic strip test for serum asialo α1-acid glycoprotein in patients with hepatic disease

Serum asialoglycoprotein (desialylated glycoproteins) concentrations have been reported to be elevated in patients with hepatic disease as compared with that of normal subjects. We recently developed a solid-phase sandwich assay for asialo α1-acid glycoprotein (AsAGP) as a representative of the serum asialoglycoproteins and evaluated the utility of this AsAGP as a diagnostic marker for liver cirrhosis (LC) and/or hepatocellular carcinoma (HCC). In this study, we developed a rapid, one-step immunochromatographic strip capable of specifically detecting AsAGP in serum specimens. We have produced a monoclonal antibody (mAb) to AGP, and based on ELISA and Western blot analysis, we have selected four hybridoma clones which generated mAbs to recognize AsAGP. In the immunochromatographic strip test, one mAb was used for conjugation with colloidal gold microparticles. Ricinus communis agglutinin (RCA) was immobilized onto a nitrocellulose membrane strip to form a result line in the path of chromatographic migration. Likewise, a control line was created above the result line by the immobilization of anti-mouse IgG. A serum specimen was then applied to the sample pad. The AsAGP in the sample specifically bound to the microparticles via mAb (As16.89) and co-migrated upward until the AsAGP was sandwiched with the immobilized lectin (RCA), revealing a visible result line. The colloidal gold microparticles without bound AsAGP continued to migrate, forming a visible control line. Thus, an AsAGP-positive specimen (> 1.5 μg/mL) yielded a result line and a control line, whereas an AsAGP-negative specimen (< 1.5 μg/mL) produced only a single control line. The entire test procedure was completed in less than 5 min. In order to examine the reliability of the testing procedures, we carried out the immunochromatographic strip test with 102 serum samples and compared the results of these tests with those obtained by ELISA. The two methods showed excellent correlation, with 83–100% above/below the cut-off value (1.5 μg/mL). Therefore, we concluded that the results of the immunochromatographic test are in excellent accordance with those of the sandwich ELISA.

Deflection and stability of membrane structures under electrostatic and Casimir forces in microelectromechanical systems

The deflection and stability of microfabricated rectangular membrane strips driven electrostatically are analyzed theoretically in this paper with the attractive Casimir force between the conducting surfaces under consideration. The analytical results of the deflection of the membrane strip under the combined electrostatic and Casimir forces show that the effect of the Casimir force on the deflection is dependent on the values of the initial gap between the membrane and the substrate w0, the applied voltage V and the fourth power of span to thickness ratio L4/h. The Casimir effect on the membrane system is dominant for smaller gap w0 and lower voltage V, yet it decreases with the gap width and the voltage increasing. When the value of w0 and V is large enough, it is feasible to neglect the Casimir force. The Casimir effect on the deflection increases with the increasing L4/H as well. The static stability of the membrane structure can be determined by a dimensionless parameter K related to the geometrical parameters of the membrane structure. The critical value KC, over which the system will become unstable and the membrane strip will collapse to the substrate, is solved through numerical calculations, and it is found that an electrostatically driven membrane structure can be designed to avoid the potential instability with a high aspect ratio (L/h).

Development of colloidal gold-based flow-through and lateral-flow immunoassays for the rapid detection of the insecticide carbaryl

One-step membrane-based competitive colloidal gold-based immunoassays in flow-through and lateral-flow formats for the rapid detection of carbaryl were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and carbaryl hapten-OVA conjugate (test line). Anti-carbaryl antibody labeled with colloidal gold particles was firstly incubated with carbaryl. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for flow-through and lateral flow of 50 and l00 μg/L, respectively. The assay time for both tests was less than 5 min, suitable for rapid testing on-site.

Substrate Binding Stoichiometry and Kinetics of the Norepinephrine Transporter

The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768-9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 micros. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP+ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP+/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates.

Ion‐Sink Phosphorus Extraction Methods Applied on 24 Soils from the Continental USA

Ion‐sink methods, such as resin membranes or FeO coated strips or filter papers, have been used as soil tests for plant available P. We have standardized a resin procedure and compared extractable soil P with the FeO method for 24 soils from the continental USA. Bray‐1 extractable P ranged from 0.3 to 221 mg kg−1 and Olsen's extractable P ranged from 1.4 to 131 mg kg−1 across all soils. We used anion resin membrane strips (RS) and traditional loose resin (LR) to extract soil P. Total surface area of one RS was 17.3 cm2, compared with 47.5 cm2 for one FeO‐coated filter paper. We used one, two, or three RS saturated with bicarbonate (RSbic) or chloride (RSCl), along with 1.5 g of moist LR saturated with either bicarbonate (LRBic) or chloride (LRCl). Using LRBic, the mean P was 64.3 mg kg−1 compared with 52.5 with one RSBic, 55.2 with two RSBic, and 63.9 with three RSBic Using LRCl, the mean P was 63.2 mg P kg−1 compared with 48.7 with one RSCl, 54.8 with two RSCl, and 57.3 with three RSCl The mean FeO‐P was 36.4 mg kg−1 Resin (Cl) hardly influenced pH of the extracting solution, LRBic and RSBic influenced pH most, and FeO‐strips had an intermediate effect on solution pH. This study shows that RS with a total surface area of 51.8 cm2 can be used to extract soil P in place of more time‐consuming LR methods.

Spectroelectrochemical Microscopy: Spatially Resolved Spectroelectrochemistry of Carrier-Based Ion-Selective Membranes

High-resolution spectroscopic imaging of the cross section of ion-selective membranes and the adjoining solution phases during real-time electrochemical measurement is termed as spectroelectrochemical microscopy (SpECM). The novel SpECM instrument utilizes wavelength-dispersive multispectral imaging of a thin membrane strip separating the two sides of a four-electrode thin-layer electrochemical cell. SpECM is aimed as a tool for optimizing the experimental conditions in mass transport-controlled ion-selective electrode membranes for improved detection limit. Some of the capabilities of the new technique are demonstrated using fix site, chromoionophore-based, pH-sensitive membranes as model systems. The experimental results are discussed in the light of the existing theory of fixed-site membranes. The quantitative expression for the time-dependent change of the free ionophore concentration across the ion-selective membrane showed close correlation to the recorded concentration profiles.

Stress-adapted numerical form finding of pre-stressed surfaces by the updated reference strategy

In this paper we present the updated reference strategy for numerical formfinding of pre-stressed membranes, which is based on standard finite elementdiscretization. The singularities of the inverse problem are regularizedby a homotopy mapping. A projection scheme is proposed where anisotropicpre-stress is defined with respect to an additional reference plane, whichreflects the initially developable surface of membrane strips in the productionprocess. Physically problematic combinations of edge geometry and surfacestress are solved by a self-adaptive stress correction scheme. The algorithmis based on a local criterion derived from differential geometry. Severalexamples illustrate the success of each idea and implementation.

Pi-Paper as an Evaluation Method of Available Phosphorus in Gypsum-Calcareous Soils

The iron oxide-impregnated paper strip (Pi test), anion resin membrane strip and Olsen (NaHCO3) tests were used to assess available phosphorus (P) in four Syrian calcareous soils treated with 0 and 190 mg P kg−1 as diammonium phosphate (DAP) or monocalcium phosphate (MCP) in an incubation study at 25°C for 0, 1, 7, 21, 42, and 72 days. The amounts of available P extracted by the three methods decreased with time of incubation, especially during the first 7 days. In general, more available P was extracted from the soils treated with MCP than that with DAP. In the three soils that don’t contain CaSO4, the available P decreased with the increasing amount of CaCO3 in the soils. Pi-P correlated well with Olsen-P or resin-P in these three soils. In the other soil, which contains 30–40% CaSO4, the amounts of Olsen-P and resin-P extracted from DAP and MCP were much less than Pi-P. The amounts of Olsen-P and resin-P in this soil were also less than that in the other three soils. It was found that of Olsen and resin- react with CaSO4 and precipitated CaCO3 that adsorbed or precipitated water-soluble P of DAP or MCP, and thus Olsen and resin-HCO3 significantly underestimated available P in the soil containing CaSO4.

Comparaison de deux techniques d’hybridation moléculaire dans l’identification de mycobactéries en pratique courante

Aim of the study. – To evaluate the performance of two commercial methods for identification of Mycobacterium species: InnoLiPA Mycobacteria first version (Innogenetics) versus Genotype MTBC and Genotype Mycobacterium (HAIN) on, respectively, 2123 and 2164 distinct isolates. Materials and methods. – Both techniques are based on the reverse hybridization of PCR products to their complementary probes immobilized on membrane strips. The InnoLiPA assay targets the 16S–23S rRNA spacer region. The HAIN test is composed of two kits: Genotype MTBC, for identification of tuberculosis complex mycobacteria, is based on gyrB DNA sequence polymorphism. Genotype Mycobacterium kit targets the 23S rDNA for identification of mycobacteria other than tuberculosis (MOTT) and tuberculosis complex mycobacteria. Both assays identify complex tuberculosis mycobacteria and respectively, eight and 12 species of MOTT. Moreover, the Genotype MTBC allows species differentiation within the M. tuberculosis complex. Results. – Eighty-eight percent and 95% of mycobacteria were identified by InnoLiPA and HAIN, respectively. Hybridization remained negative for 11% of isolates with InnoLiPA and 4% with HAIN. An identification of MOTT was obtained by conventional identification in all cases after the use of InnoLiPA. MOTT and one M. tuberculosis was obtained after HAIN procedure. Unidentified species were complementary to a specific probe in 5% of the cases with InnoLiPA and 17% with HAIN. Conclusion. – HAIN identifies more mycobacteria species than does InnoLiPA and allows identification in the M. tuberculosis complex. However, failure in identification occurs only with MOTT with InnoLiPA when one M. tuberculosis was found among mycobacteria non identified with HAIN.

Antimicrobial resistance of Campylobacter jejuni and C coli isolates in Japan

Campylobacter jejuni and Campylobacter coli are foodborne enteric pathogens of human beings (Allos 2001), with poultry meat being a major source of Campylobacter infection (Park 2002). Recently, increasing levels of resistance in C jejuni and C coli to macrolide and fluoroquinolone antibiotics have become a worldwide problem as these are the therapeutic agents used for Campylobacter infections in humans (Aarestrup and Engberg 2001). The aims of this study were to investigate the antimicrobial resistance of Campylobacter isolates from human beings and poultry meat in Kanagawa, Japan, and to detect point mutations related to macrolide and quinolone resistance by using a line probe assay (MQ-LiPA), designed for the rapid and easy simultaneous detection of macrolide resistance mutations in 23S rDNA and quinolone resistance mutations in gyrA (Niwa and others 2003). In total, 249 isolates were used in this study: 193 human Campylobacter strains and 56 poultry meat strains were isolated in Kanagawa prefecture from 1979 to 2001 and from 1997 to 2000, respectively, and identified by biological and biochemical tests in the Kanagawa Prefectural Public Health Laboratory. All strains were cultured on Mueller-Hinton (MH) agar plates (Oxoid) at 42°C under microaerobic conditions (85 per cent nitrogen, 5 per cent oxygen, 10 per cent carbon dioxide) for 24 hours. The following antimicrobial agents were tested: nalidixic acid (Sigma), ofloxacin (Sigma), erythromycin (Sigma), tetracycline (Wako), ampicillin (Sigma), gentamicin (Sigma) and phosphomycin (Wako). Minimum inhibitory concentrations (MICs) were determined by an agar dilution method using MH agar plates. All MIC plates were inoculated with approximately 104 colony-forming units of each strain per spot using a multipoint inoculator. A control plate without antibiotics was also incubated at the end of each procedure. The plates were incubated for 24 hours at 42°C under microaerobic conditions. The MIC was defined as the lowest concentration producing no visible growth. The breakpoints were used according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS 2002) except for ofloxacin and phosphomycin. No criteria were shown for ofloxacin or phosphomycin in the NCCLS document, so a breakpoint of 8 mg/litre was used for ofloxacin (Sanchez and others 1994) and 128 mg/litre for phosphomycin (Gomez-Garces and others 1995). The line probe assay (MQ-LiPA) was performed by the method described by Niwa and others (2003). This method is based on PCR and reverse hybridisation to a number of oligonucleotide probes immobilised as parallel lines on a nylon membrane strip. Oligonucleotide probes were tailed with dTTP (Gibco BRL). Poly (dT)-tailed probes and biotinylated DNA used as a colour development control were immobilised as parallel lines on the nylon membrane strip by ultraviolet exposure. The PCR was performed using primers designed according to the sequences of 23S rDNA and gyrA. The results of the antimicrobial susceptibility tests for the C jejuni and C coli isolates are shown in Table 1. In human isolates, the frequency of strains resistant to the quinolones, nalidixic acid and ofloxacin, increased remarkably, while tetracycline-resistant strains decreased during the study periods 1979 to 1989 and 1990 to 2001. Quinolone-resistant Campylobacter strains were observed from 1977 in both human and poultry meat isolates in this study. In Japan, many kinds of fluoroquinolones were introduced from the second half of the 1980s and the first half of the 1990s in the medical and veterinary fields, respectively. Poultry meat is a main source of Campylobacter infection in Japan (Ono and Yamamoto 1999) and various other countries (Padungton and Kaneene 2003), and the increase in the incidence of fluoroquinolone resistance in human isolates appears to be related to that in poultry. There was a marked decrease in erythromycin-resistant strains in human C jejuni isolates between the two study periods (1979 to 1989 and 1990 to 2000). The resistance level of all erythromycin-resistant C jejuni strains was low (MIC 8 to 16 mg/litre), while high-level (MIC ≥128 mg/litre) erythromycin-resistant strains were observed only in C coli. Low-level erythromycin-resistant strains might not be truly resistant, because the amount of carbon dioxide in the atmosphere increases the MICs of erythromycin (Rautelin and others 1991). Range of MIC (mg/litre) Profile by MQ-LiPA* Nalidixic acid Ofloxacin Erythromycin

Disposable noncompetitive immunosensor for free and total prostate-specific antigen based on capacitance measurement.

This work reports on the successful integration of a one-step lateral flow immunoassay format and impedance detection of the specific affinity event using an electrochemical transducer coated with a pH-sensitive polymer layer. This approach was particularly applied to the development of a rapid single-use immunosensor for the sensitive detection of free and total prostate-specific antigen (f-PSA, t-PSA) tumor marker. Strips of nitrocellulose membrane were coated with appropriate antibodies to f-PSA and t-PSA and used as solid supports for the performance of noncompetitive immunoassays where PSA was allowed to react with both immobilized anti-PSA antibody and anti-PSA urease enzyme conjugate for less than 1 min. An additional piece within the device consisting of a storage blister filled with a urea solution allowed the rapid washing of unbound species from the membrane strips and simultaneous urea hydrolysis catalyzed by the bound urease conjugate in an automatic fashion. The hydrolysis of urea increased the pH of the reaction media, which in turn induced a breakdown of the polymer layer on the transducer and a consequent measurable change in capacitance of the system. This was easily recorded at a given frequency over a 30-min period. Overall, we describe a one-step immunosensor prototype that exhibits enough sensitivity to detect both forms of PSA at concentration levels down to 3 ng/mL. With the possibility of being portable and considering its ease of use, robustness, and simplicity, this device has great potential as a tool for the screening and early detection of prostate cancer.

Multi-analyte single-membrane biosensor for the serotype-specific detection of Dengue virus.

A multi-analyte biosensor based on nucleic acid hybridization and liposome signal amplification was developed for the rapid serotype-specific detection of Dengue virus. After RNA amplification, detection of Dengue virus specific serotypes can be accomplished using a single analysis within 25 min. The multi-analyte biosensor is based on single-analyte assays (see Baeumner et al (2002) Anal Chem 74:1442-1448) developed earlier in which four analyses were required for specific serotype identification of Dengue virus samples. The multi-analyte biosensor employs generic and serotype-specific DNA probes, which hybridize with Dengue RNA that is amplified by the isothermal nucleic acid sequence based amplification (NASBA) reaction. The generic probe (reporter probe) is coupled to dye-entrapping liposomes and can hybridize to all four Dengue serotypes, while the serotype-specific probes (capture probes) are immobilized through biotin-streptavidin interaction on the surface of a polyethersulfone membrane strip in separate locations. A mixture of amplified Dengue virus RNA sequences and liposomes is applied to the membrane and allowed to migrate up along the test strip. After the liposome-target sequence complexes hybridize to the specific probes immobilized in the capture zones of the membrane strip, the Dengue serotype present in the sample can be determined. The amount of liposomes immobilized in the various capture zones directly correlates to the amount of viral RNA in the sample and can be quantified by a portable reflectometer. The specific arrangement of the capture zones and the use of unlabeled oligonucleotides (cold probes) enabled us to dramatically reduce the cross-reactivity of Dengue virus serotypes. Therefore, a single biosensor can be used to detect the exact Dengue serotype present in the sample. In addition, the biosensor can simultaneously detect two serotypes and so it is useful for the identification of possible concurrent infections found in clinical samples. The various biosensor components have been optimized with respect to specificity and sensitivity, and the system has been ultimately tested using blind coded samples. The biosensor demonstrated 92% reliability in Dengue serotype determination. Following isothermal amplification of the target sequences, the biosensor had a detection limit of 50 RNA molecules for serotype 2, 500 RNA molecules for serotypes 3 and 4, and 50,000 molecules for serotype 1. The multi-analyte biosensor is portable, inexpensive, and very easy to use and represents an alternative to current detection methods coupled with nucleic acid amplification reactions such as electrochemiluminescence, or those based on more expensive and time consuming methods such as ELISA or tissue culture.

Insulin Receptor Substrate-2-dependent Interleukin-4 Signaling in Macrophages Is Impaired in Two Models of Type 2 Diabetes Mellitus

We have shown previously that hyperinsulinemia inhibits interferon-alpha-dependent activation of phosphatidylinositol 3-kinase (PI3-kinase) through mammalian target of rapamycin (mTOR)-induced serine phosphorylation of insulin receptor substrate (IRS)-1. Here we report that chronic insulin and high glucose synergistically inhibit interleukin (IL)-4-dependent activation of PI3-kinase in macrophages via the mTOR pathway. Resident peritoneal macrophages (PerMPhis) from diabetic (db/db) mice showed a 44% reduction in IRS-2-associated PI3-kinase activity stimulated by IL-4 compared with PerMPhis from heterozygote (db/+) control mice. IRS-2 from db/db mouse PerMPhis also showed a 78% increase in Ser/Thr-Pro motif phosphorylation without a difference in IRS-2 mass. To investigate the mechanism of this PI3-kinase inhibition, 12-O-tetradecanoylphorbol-13-acetate-matured U937 cells were treated chronically with insulin (1 nm, 18 h) and high glucose (4.5 g/liter, 48 h). In these cells, IL-4-stimulated IRS-2-associated PI3-kinase activity was reduced by 37.5%. Importantly, chronic insulin or high glucose alone did not impact IL-4-activated IRS-2-associated PI3-kinase. Chronic insulin + high glucose did reduce IL-4-dependent IRS-2 tyrosine phosphorylation and p85 association by 54 and 37%, respectively, but did not effect IL-4-activated JAK/STAT signaling. When IRS-2 Ser/Thr-Pro motif phosphorylation was examined, chronic insulin + high glucose resulted in a 92% increase in IRS-2 Ser/Thr-Pro motif phosphorylation without a change in IRS-2 mass. Pretreatment of matured U937 cells with rapamycin blocked chronic insulin + high glucose-dependent IRS-2 Ser/Thr-Pro motif phosphorylation and restored IL-4-dependent IRS-2-associated PI3-kinase activity. Taken together these results indicate that IRS-2-dependent IL-4 signaling in macrophages is impaired in models of type 2 diabetes mellitus through a mechanism that relies on insulin/glucose-dependent Ser/Thr-Pro motif serine phosphorylation mediated by the mTOR pathway.

Membrane stripping versus single dose intracervical prostaglandin gel administration for cervical ripening

Membrane stripping versus single dose intracervical prostaglandin gel administration for cervical ripening

Cervical ripening methods for labor induction

The indication for labor induction has been increasing in the world. It is known that cervical conditions are directly associated to the success of labor induction. Knowledge of cervix anatomy and physiology during pregnancy and of the different methods for cervical ripening is essential for indicating the best cervical ripening method in a given situation, therefore obtaining the best outcomes following labor induction. This is a challenge for obstetricians where not every method is readily available and accessible and C-sections rates are very high as in Brazil. Some methods are discussed in this paper including breast stimulation, membrane stripping, and the use of relaxin, oxytocin, prostaglandins, hyaluronidase, mifepristone, laminaria and Foley catheter.

Association of Vasa Previa at Delivery With a History of Second-Trimester Placenta Previa

Vasa previa is a rare obstetric disorder characterized by velamentous insertion of the fetal vessels over the mouth of the cervix. Reported fetal mortality estimates range from 58% to 73%. Ultrasonography has proved helpful for diagnosing this condition prenatally, improving outcomes, but little is known about any antecedent conditions that predispose to vasa previa. This study evaluated 1 possible factor, a history of second-trimester placenta previa, in 13 women presenting with vasa previa in the years 1991 through 2001. Deliveries totalled nearly 73,000 during this period. Vasa previa occurred once in every 5614 deliveries. Four control women with normal placentation were matched with each study patient. Eight of the 13 cases were detected in the third trimester by demonstrating fetal blood flow over the cervical os using pulsed color flow Doppler ultrasonography. Two other women presented at the time of amniotomy during labor with acute vaginal bleeding and subsequent fetal bradycardia. In 2 cases, the fetal vessels were palpated within the amniotic membranes during labor. One case was diagnosed after delivery, the fetus having died; torn fetal vessels were found when viewing the placenta. This woman had undergone membrane stripping during a prenatal visit at 41 weeks gestation. Three of the 13 women with vasa previa were nulliparous. Demographic features were similar in the study and control groups, although affected women tended to be older and to have undergone dilation and curettage more often than control women. Antepartum vaginal bleeding, cesarean delivery, and cesarean hysterectomy all were more frequent in study women. Two women, including the 2 with a dead fetus, were delivered vaginally. Nine of 13 women (69.2%) had placenta previa diagnosed by midtrimester ultrasonography. This was true for only 2 control women (3.8%), making the association highly significant. This retrospective study disclosed a statistically impressive relationship between vasa previa at delivery and a history of second-trimester placenta previa. Prospective studies of women with the latter condition would be of interest.

Nafion membrane electrophoresis with direct and simplified end-column pulse electrochemical detection of amino acids.

A novel electrophoresis technique, in which the separation column was replaced by a strip of Nafion membrane (5.0 cm x 0.20 mm x 0.25 mm), was developed for the separation of an amino acid mixture (glycine, asparic acid and lysine), followed by quadruple-pulse electrochemical detection. Nafion membrane contains hydrophilic pores (10-20 A and 50-60 A in size) acting as very narrow electrophoresis channels. The fixed-charge sites (-SO(3) (-)) on the hydrophilic pore surface provide a strong charged background. A platinum disk electrode (0.90 mm inner diameter) was employed as the detection electrode and the electrophoresis cathode was used as the quasi-reference and counter electrode for the end-column electrochemical detector, without decoupler. Under optimized conditions the mixture of amino acids could be separated at a voltage of only 90 V with a detection limit of 10(-7) M, indicating that Nafion membrane electrophoresis is a potentially attractive technique for the separation of small organic molecules or ions.