Use of an albumin-binding domain for the selective immobilisation of recombinant capture antibody fragments on ELISA plates

Human embryonic kidney cells (HEK 293) are widely used as an expression system in studies of ion channels. However, their endogenous ionic currents remain largely unidentified. To characterize these currents, we performed patch clamp experiments on this expression system. In whole-cell voltage clamp mode, the HEK 293 cells showed mainly outward currents using physiological concentrations of Na+ and K+ and symmetric concentrations of Cl- (150 mM) across the plasma membranes. K+ currents contributed to a small portion of these outward currents, since a shift of the reversal potentials of only approximately 20 mV was seen with a change of extracellular K+ concentration from 3 to 150 mM. In contrast, the reversal potential shifted approximately 25 mV when extracellular Cl- was reduced to 50 mM, indicating that most of the outward currents are carried by Cl-. In inside-out patches, several distinct Cl- currents were identified. They were: (1) 350 pS Cl- current, which was voltage-activated and had a moderate outward rectification; (2) 240 pS Cl- current with a weak outward rectification; and (3) 55 pS Cl- current, which was voltage-activated, sensitive to DIDS, and showed a strong outward rectification. Activation of these Cl- currents did not require an elevation of free Ca2+ level in the cytosol. Besides these three currents, we observed two other Cl- currents with much smaller conductances (25 and 16 pS, respectively). Two different K+ currents were seen in the HEK 293 cells, with one of them (125 pS) showing inward rectification and the other (70 pS) outward rectification. Moreover, a 50 pS cation channel was recorded in these cells. The presence of a variety of ion channels in the HEK 293 cells suggests that a great precaution needs to be taken when this expression system is used in studies of several similar ion channels.     

Becoming a clinical psychologist in the United Kingdom.

The process by which one becomes a qualified clinical psychologist in the United Kingdom (U.K.) is described so that American (United States) clinical psychologists visiting the U.K. may better understand the context in which their British counterparts work. The process begins with the admissions criteria of training programs and ends with one's acceptance as a fully qualified, independent clinical psychologist. Educational and health care issues are described as factors relevant in shaping the structure of clinical psychology programs. Advantages and disadvantages of the British system are discussed in the light of continuing political changes, and some suggestions for improvements are made. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A Study of equilibria in the Fe-Cr-Ni-Mo-C-N system at 1273 K

The phase equilibria in the Fe-rich corner of the Fe-Cr-Ni-Mo-C-N system have been studied at 1273 K using a sealed capsule technique to measure the C and N activities and the electron microprobe to measure the compositions of the individual phases following identification by X-ray analysis. Some of the new information was combined with previous assessments of the Fe-Cr-N, Fe-Ni-N, and Fe-Cr-Ni systems and a new assessment of the Cr-Ni-N system in order to assess the thermodynamic properties of the Fe-Cr-Ni-N system. A set of parameters is obtained, based mainly upon experimental information from 1273 K (1000 °C) and 1473 K (1200 °C), which can be used for calculations of the Fe-Cr-Ni-N phase diagram in this temperature range. Isothermal sections are presented and show reasonable agreement with experimental data not used in the assessment. The thermodynamic analysis is restricted to the Fe-Cr-Ni-N system, but some experimental data are also presented for alloys containing Mo and C. Formerly with Royal Institute of Technology, Stockholm.     

The surface behavior of mercury on iron systems

Thermal desorption Auger electron spectroscopy (TDAES) was used to investigate the circumstances under which mercury is adsorbed on the surface of iron in the temperature interval of 85 to 298 K. The effects of chlorine and oxygen modifications on the iron surface have also been investigated within the same temperature interval. It was seen that chlorine reduced the adsorption of mercury on polycrystalline iron at 85 K, as did oxygen. On the clean iron system at 298 K, only one monolayer (ML) of mercury adsorbed. The physisorption of mercury on chlorine and oxygen layers at low temperatures (LTs) is discussed in combination with the calculated activation energies of desorption, as well as the factors affecting the mechanism of adsorption at room temperature.     

Soil Water Retention and Conductivity When Vapor Flow Is Important

Models of soil water transport often calculate conductivity K from the water retention curve (WRC). Residual water content (θr) has been defined as θ where K = 0. When nonisothermal, coupled vapor and liquid water transport are considered, θr>0 fails because vapor transport often reduces θ to near zero. The author’s objective was to test a model that used unsaturated K(θ) with θ dependence typical of θr>0, while a WRC with θr = 0 was used elsewhere in the model. The system was a closed column of steady state, unsaturated, nonisothermal fine quartz sand with temperature (T) ranging from 5 to 40°C. Soil parameters were adjusted to simulate replicated experimental data from one initial θ condition. The model predicted θ and T within the range of the experimental data and reproduced the sharp drying front. It also satisfactorily modeled experiments with several different initial θ. Model heat flux predictions averaged 11% more than measured values. Experiments performed with two soil column lengths were not substantially different.     

Identification of a peptide inducing experimental autoimmune uveoretinitis (EAU) in H-2Ak-carrying mice

When certain strains of mice bearing H-2Ak are immunized with the interphotoreceptor retinoid-binding protein (IRBP), EAU is induced. Thus far uveitogenic determinant(s) has not been determined in the H-2Ak mouse system. In addition it is hard to prepare purified IRBP. In the present study, to circumvent these problems we attempted to identify uveitogenic peptides derived from bovine IRBP in H-2Ak haplotype mice. Six peptides which had been selected according to the H-2Ak binding motif (Dxxxxxxxx[A, R, T]) were synthesized. We report here that all the peptides are immunogenic but only one peptide, K2, which consisted of IRBP201-216 residues, induces EAU in various mice carrying H-2Ak. Amino acid substitution of K2 revealed that the core region interacted with both H-2Ak and T cell antigen receptor (TCR). The amino acid sequence of the core region derived from bovine IRBP was identical to the corresponding region of mouse IRBP. In addition, K2 appeared to be a natural peptide antigen processed from bovine IRBP. Altogether, we concluded that K2 is one of the natural autoantigens involved in induction of EAU in H-2Ak mice.     

Cytomegalovirus-induced interstitial pneumonitis in a patient with systemic lupus erythematosus

BACKGROUND: Antibodies of the Knops system have been referred to as nonneutralizable because they cannot be inhibited with serum, saliva, or urine. Because the Knops system antigens have been located on complement receptor 1 (CR1), the question of whether the antibodies could be neutralized with soluble CR1 (sCR1) produced by recombinant DNA techniques was studied. STUDY DESIGN AND METHODS: First, radiolabeled immunoprecipitation techniques were used to test sCR1 for the expression of the high-incidence Knops system antigens. Then, a total of 45 antibodies were neutralized with sCR1, including the following: one each of anti-Cr(a), -Dr(a), -Do(b), -Hy, -Ge, -Jr(a), -Sc1, -Jk(a), -Cs(a), and -Kp(b); two each of anti-Lu(b), -Yt(a), and -JMH; three each of anti-McC(a), -Rg, and -Sl(a); and four each of anti-Ch, -Kn(a), -Yk(a), -Kn/McC. In addition, two examples of anti-Kn(a) + K, one example of anti-Sl(a) + K + Fy(a), and one example of anti-Yk(a) + E were tested. The sCR1 was added to each test serum and 6-percent albumin was added to the control; this was followed by neutralization incubation for 5 minutes at 25 degrees C. The antibody samples were then tested by a low-ionic-strength solution, anti-human globulin technique. RESULTS: The sCR1 expressed Kn(a), McC(a), Sl,a and Yk(a). All Knops system antibodies (n = 22) were neutralized by the sCR1, but none of the other 23 alloantibodies decreased in reactivity. The samples containing antibodies of two specificities showed inhibition of the Knops system antibody but not of the second antibody. CONCLUSION: This neutralization method, in which recombinant protein is used, provides an expedient and definitive method of identifying Knops system antibodies.     

A developmentally regulated kinesin-related motor protein from Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive alpha-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end-directed microtubule motility in vitro at a speed of 0.14 micron/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells.     

Investigation into the equilibrium of the wustite and spinel solutions in the Fe-Ge-O system

Equilibrium conditions of the wustite and spinel solutions in the Fe-Ge-O system in temperature range T = 1100–1300 K are investigated by measuring the emf of the galvanic element with solid electrolyte (the emf method). X-ray diffractometry and electron probe microanalysis were used to determine the phase composition. It is established that the iron magnetite and germanate are partially miscible in one another. The isothermal sections of the phase diagram of the Fe-Fe₃O₄-Fe₂GeO₄ system at T = 1273 K and 1173 K are constructed.     

Specific induction of hepatocellular adenomas by transplacental administration of ENU in the tsc2 gene mutant (Eker) rat

Recombinant human m-calpain was produced in a soluble form at a level of 20 mg/liter of Sf-9 cell culture by the coexpression of recombinant human m-calpain large (m80K) and small (30K) subunits using a baculovirus expression system. The expressed m-calpain was purified by sequential column chromatographies on DEAE-Toyopearl, gel-filtration, and Mono Q by the same method used to purify native m-calpain. The recombinant m-calpain had a specific activity of 691 U/mg and a Ka value (Ca2+ requirement for 50% caseinolysis activity) of 0.4 mM, which are essentially identical to those of native rabbit m-calpain. A mutant m-calpain large subunit, m-C105S-80K, where the active-site cysteine-105 is converted to serine by site-directed mutagenesis, was coexpressed with 30K in Sf-9 cells, purified, and characterized. m-C105S-calpain does not degrade casein nor an artificial tetra-peptide substrate, succinyl-Leu-Leu-Val-Tyr-MCA. Further, it shows no autolytic activity with Ca2+. This is the first report of the large-scale production of a fully active m-calpain species in the baculovirus system.     

Neighborhood effects using a partial priming methodology: Guessing or activation?

K. Pugh, K. Rexer, M. Peter, and L. Katz (1994) found longer lexical-decision latencies to 4-letter words when an ambiguous letter (one from which neighbors could be formed) was delayed than when an unambiguous letter (one from which no neighbors could be formed) was delayed. They suggested that this was due to competition between partially activated words. However, K. I. Forster and D. Shen (1996) suggested that this effect may be due to participants' generating hypotheses on the basis of the previewed trigram. The authors conducted 2 experiments that used a partial priming methodology and found that lexical decision latencies were longer to words preceded by ambiguous trigrams than unambiguous trigrams when (a) the target was the highest frequency member in its neighborhood and (b) the prime was masked and presented for 60 ms. These results are inconsistent with Forster and Shen's prediction of no effect of prime ambiguity under these conditions, and they indicate that the ambiguity effect was not due to hypothesis generation on the basis of the partial primes. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Characterisation of strain-specific sequences from an abortifacient strain of ovine Chlamydia psittaci using subtraction hybridisation

BACKGROUND: The antigens of the human Rh system are of great clinical significance in transfusion medicine and pregnancy. Of the Rh system antigens, D is clinically the most important, being one of the most immunogenic structures arising from human cells. The human D antigen represents a collection of epitopes expressed on a red cell membrane protein that is predicted to have 12 membrane-spanning segments giving rise to six exofacial domains. STUDY DESIGN AND METHODS: By site-directed mutagenesis using the method of inverse polymerase chain reaction, cE and D cDNA mutant constructs were generated with changes to the RHD-specific residues 350, 353, and 354 in the predicted sixth exofacial loop. Each mutant cDNA was subcloned into the pBabe puromycin retroviral vector, and supernatants were used to transduce K562 cells. Puromycin-resistant K562 clones were screened by flow cytometric analysis using a panel of monoclonal antibodies with specificities to ep (epitope) D1 through epD9. RESULTS: De novo expression of epD3 and epD9 was generated in the K562 cell lines expressing the mutated cE polypeptide (cE-Asp350His, Gly353Trp, Ala354Asn). Expression of c and E was unaffected. Conversely, the cells expressing the mutated D polypeptide demonstrated loss of expression of epD1, epD2, epD3, epD4, and epD9. CONCLUSION: The data provide strong evidence for the critical involvement of three amino acids, Asp350, Gly353, and Ala354, in the expression of epD3 and epD9 on the predicted sixth external domain of the D protein. This domain also appears to be essential for the expression of epD1, epD2, and epD4, as a loss of expression of these epitopes was observed in K562 cells transduced with the Dmut construct (encoding His350, Trp353, and Asn354). The K562/Dmut cell line has an identical molecular and serologic profile as the red cell D(IVb) phenotype, which confirms that retroviral gene transfer of Rh cDNA into K562 cells provides us with a powerful means by which to further map epitopes of D.     

Intracavitary afterloading therapy as a new technique of boost irradiation in anal canal carcinoma

PURPOSE: There are different techniques of boost irradiation in the treatment of patients with anal carcinoma. A new system of applicators is presented, which can be used for an intracavitary afterloading therapy. MATERIAL AND METHODS: Three different applicators are available, the first with a central catheter (K1), a second with 5 semicircular fixed catheters (K2) and an eccentric shield, a third with 8 circular fixed catheters and a central shield (K3). RESULTS: The adequate choice of applicator and catheters takes into consideration the individual localisation and extension of anal carcinoma in planning therapy. Thus, in circular growing tumors, an irradiation of the whole circumference of the anal canal is possible. In non-circular growing tumors, the dose applied in the non-affected part of the anal canal can be reduced to a quarter of the dose applied at the tumor. CONCLUSION: The new system of intracavitary afterloading therapy is a good alternative to previous techniques of boost irradiation in the treatment of anal carcinoma. By means of this technique, irradiation can be highly individualized, the tumor better included and non-affected sections of the anal canal saved.     

Marriage season, promptness of successful pregnancy and first-born sex ratio in a historical natural fertility population--evidence for sex-dependent early pregnancy loss?

Interleukins are potent intercellular messenger peptides, initially found in cells of the immune system and best known for producing chronic, genomic effects in target cells. Here, interleukin-1beta (IL-1beta) was tested for acute effects on neurotransmitter release. The human neuroblastoma-derived cell-line SH-SY5Y is a model for mature post-ganglionic sympathetic neurones and release of tritiated noradrenaline from these cells was measured, in response to stimulation with either elevated extracellular K+ concentration (100 K+) orveratridine. Pre-incubation for 15-25 min with 60 pM (but not 0.06 pM) IL-1beta significantly reduced 100 K+-evoked release (by approximately 75%). The interleukin was without effect on basal or veratridine-evoked noradrenaline release. The present data suggest two distinct stimulatory pathways: one that is activated by 100 K+ and veratridine and is unaffected by IL-1beta and another that is activated by 100 K+ but not veratridine and is inhibited by IL-1beta. The acute depression of 100 K+-evoked transmitter release may be involved in immune system-nervous system interactions.     

Two functionally different Na/K pumps in cardiac ventricular myocytes

The whole-cell patch-clamp technique was used to voltage clamp acutely isolated myocytes at -60 mV and study effects of ionic environment on Na/K pump activity. In quiescent guinea pig myocytes, normal intracellular Na+ is approximately 6 mM, which gives a total pump current of 0.25 +/- 0.09 pA/pF, and an inward background sodium current of 0.75 +/- 0.26 pA/pF. The average capacitance of a cell is 189 +/- 61 pF. Our main conclusion is the total Na/K pump current comprises currents from two different types of pumps, whose functional responses to the extracellular environment are different. Pump current was reversibly blocked with two affinities by extracellular dihydro-ouabain (DHO). We determined dissociation constants of 72 microM for low affinity (type-1) pumps and 0.75 microM for high affinity (type-h) pumps. These dissociation constants did not detectably change with two intracellular Na+ concentrations, one saturating and one near half-saturating, and with two extracellular K+ concentrations of 4.6 and 1.0 mM. Ion effects on type-h pumps were therefore measured using 5 microM DHO and on total pump current using 1 mM DHO. Extracellular K+ half-maximally activated the type-h pumps at 0.4 mM and the type-1 at 3.7 mM. Extracellular H+ blocked the type-1 pumps with half-maximal blockade at a pH of 7.71 whereas the type-h pumps were insensitive to extracellular pH. Both types of pumps responded similarly to changes in intracellular-Na+, with 9.6 mM causing half-maximal activation. Neither changes in intracellular pH between 6.0 and 7.2, nor concentrations of intracellular K+ of 140 mM or below, had any effect on either type of pump. The lack of any effect of intracellular K+ suggests the dissociation constants are in the molar range so this step in the pump cycle is not rate limiting under normal physiological conditions. Changes in intracellular-Na+ did not affect the half-maximal activation by extracellular K+, and vice versa. We found DHO-blockade of Na/K pump current in canine ventricular myocytes also occurred with two affinities, which are very similar to those from guinea pig myocytes or rat ventricular myocytes. In contrast, isolated canine Purkinje myocytes have predominantly the type-h pumps, insofar as DHO-blockade and extracellular K+ activation are much closer to our type-h results than type-1. These observations suggest for mammalian ventricular myocytes: (a) the presence of two types of Na/K pumps may be a general property. (b) Normal physiological variations in extracellular pH and K+ are important determinants of Na/K pump current. (c) Normal physiological variations in the intracellular environment affect Na/K pump current primarily via the Na+ concentration. Lastly, Na/K pump current appears to be specifically tailored for a tissue by expression of a mix of functionally different types of pumps.     

Thermodynamics of the system iron-aluminum-oxygen between 1073 K and 1573 K

Equilibrium oxygen pressures of the univariant ternary coexistences hematite + spinel + alumina, alloy + wustite + spinel, and alloy + spinel + alumina were determined at temperatures in the range 1123 to 1423 K using solid state electrochemical cells. The compositions of alloys and oxide phases of this Fe-Al-0 system that equilibrated with one another at 1273, 1423, and 1573 K were determined. These results and those from the literature are used to determine stabilities of the ternary oxide phases as a function of oxygen pressure and composition by constructing equilibrium oxygen pressure diagrams at 1073, 1173, and 1273 K. Formerly with McMaster University     

Function of conserved tryptophans in the Aspergillus niger glucoamylase 1 starch binding domain

Nuclear magnetic resonance (NMR) and ultraviolet (UV) difference spectroscopy were used to assess the role of a number of tryptophan residues in the granular starch binding domain (SBD) of glucoamylase 1 from Aspergillus niger. Wild-type SBD and three variant (W563K, W590K, and W615K) proteins were produced using an A. niger expression system. Titration studies were conducted with beta-cyclodextrin (betaCD), a cyclic analogue of starch, as the ligand. The NMR studies show that the W563K and W590K variants only bind 1 equiv while the wild-type protein forms a 2:1 (ligand:protein) complex. It also clearly demonstrates the abolition of binding at site 1 and site 2 in W590K and W563K, respectively. UV difference spectroscopy was used to calculate dissociation constants with addition of betaCD: 14.4 microM (apparent) for the wild type, 28.0 microM for W563K, and 6.4 microM for W590K. The implication of this is that the two binding sites have unequal contributions to the overall binding of the SBD which may be related to functional differences between the two binding sites. The low stability of the third variant, W615K, suggests that this tryptophan is not involved in binding but has an essential structural role.