High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells

CD22 (Siglec-2) is a key regulator of B cell signaling whose function is modulated by interaction with extracellular glycan ligands mediated through its N-terminal Ig domain. Its preferred ligand is the sequence Sia alpha2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins, and by binding to CD22 in cis causes CD22 to appear "masked" from binding to synthetic sialoside probes. Yet, despite the presence of cis ligands, CD22 redistributes to sites of cell contact by binding to trans ligands on neighboring cells. In this study, we demonstrate the dynamic equilibrium that exists between CD22 and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells. Consistent with the constitutive endocytosis reported for CD22, the probes are internalized once bound, demonstrating that CD22 is an endocytic receptor that can carry ligand-decorated "cargo" to intracellular compartments. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.     

Binding of 13-HODE and 15-HETE to phospholipid bilayers, albumin, and intracellular fatty acid binding proteins. implications for transmembrane and intracellular transport and for protection from lipid peroxidation

Transport and utilization of fatty acids (FA) in cells is a multistep process that includes adsorption to and movement across the plasma membrane and binding to intracellular fatty acid binding proteins (FABP) in the cytosol. We monitored the transbilayer movement of several polyunsaturated FA and oxidation products (13-hydroxy octadecadienoic acid (HODE) and 15-hydroxytetraenoic acid (HETE)) in unilamellar protein-free phospholipid vesicles containing a fluorescent pH probe. All FA diffused rapidly by the flip-flop mechanism across the model membrane, as revealed by pH changes inside the vesicle. This result suggests that FA oxidation products generated in the cell could cross the plasma or nuclear membrane spontaneously without a membrane transporter. To illuminate features of extra- and intracellular transport, the partitioning of unsaturated FA and oxidized FA between phospholipid vesicles and albumin or FABP was studied by the pyranin assay. These experiments showed that all polyunsaturated FA and oxidized FA (13-HODE and 15-HETE) desorbed rapidly from the phospholipid bilayer to bind to bovine serum albumin, which showed a slight preference for the unsaturated FA over the oxidized FA. FABP rapidly bound FA in the presence of phospholipid bilayers, with a preference of 13-HODE over the unsaturated FA and with a specificity depending on the type of FABP. Liver FABP was significantly more effective than intestinal FABP in binding 13-HODE in the presence of vesicles. The more effective binding of the FA metabolite, 13-HODE, than its precursor 18:2 by FABP may help protect cellular membranes from potential damage by monohydroxy fatty acids and may contribute a pathway for entry of 13-HODE into the nucleus.     

Time budget of caged rhesus monkeys exposed to a companion,a PVC perch,and a piece of wood for an extended time

The present investigation assesses the percentage of time that rhesus monkeys (Macaca mulatta) are kept occupied by a more complex cage environment. Sixty animals were continuously exposed for at least 1.5 years to a compatible companion for social interaction, a suspended plastic pipe for perching, and a branch segment for gnawing. The behavior of the partners of each pair was recorded for 60 minutes when the animals were not distracted by human activities. Individuals spent an average of 23.5% of the time interacting with the companion, but only 10.4% with the plastic pipe and 4.8% with the branch segment. Differences were statistically significant. Females were socially more active than males. Subadult animals (3.5–4 years) used both inanimate objects significantly more than did adult animals (9–30 years). It was concluded that (1) a compatible companion, a suspended plastic pipe, and a loose branch segment remain effective stimuli for caged rhesus monkeys after more than 1 year of exposure and (2) a compatible companion is of greater stimulatory value–particularly for adults–than are inanimate objects.     

Ionic selectivity, saturation and block in gramicidin A channels: I. Theory for the electrical properties of ion selective channels having two pairs of binding sites and multiple conductance states.

A model for the gramicidin A channel is proposed which extends existing models by adding a specific cationic binding site at each entrance to the channel. The binding of ions to these outer channel sites is assumed to shift the energy levels of the inner sites and barriers and thereby alter the channel conductance. The resulting properties are analyzed theoretically for the simplest case of two inner sites and a single energy barrier. This for-site model (two outer and two inner) predicts that the membrane potential at zero current (Uo) should be a Goldman-Hodgkin-Katz equation with concentration-dependent permeability ratios. The coefficients of the concentration-dependent terms are shown to be related to the peak energy shifts of the barrier and to the binding constants of the outer sites. The thory also predicts the channel conductance in symmetrical solutions to exhibit three limiting behaviors, from which the properties of the outer and inner sites can be characterized. In two-cation symmetrical mixtures the conductance as a function of mole fraction is shown to have a minimum, and the related phenomenon of inhibition and block exerted by one ion on the other is explained explicitly by the theory. These various phenomena, having ion interactions in a multiply occupied channel as a common physical basis, are all related (by the theory) through a set of measurable parameters describing the properties of the system.     

Solid-phase assay for the detection of low-abundance enzymes, and antibodies to enzymes in immune reactions, using acid sphingomyelinase as a model

The development of a solid-phase immunosorbent assay, suitable for use with enzyme antigens, is described. Acid sphingomyelinase and a mouse monoclonal anti-sphingomyelinase antibody have been used to determine optimal conditions for the assay. The assay involves immobilization of a second antibody (anti-mouse IgG) in the wells of a polyvinyl microtiter plate. Soluble immune complexes of first antibody (monoclonal anti-sphingomyelinase) and antigen (sphingomyelinase), incubated in separate vials, are then reacted in the anti-mouse IgG-coated assay wells, and the extent of the cross-reaction between antibody and antigen is measured by direct assay of enzyme retained in the well. A necessary condition of the assay is that antibody must not inhibit enzyme activity, which makes it especially suitable for monoclonal antibodies. The assay finds useful application in hybridoma fluid screening, equivalence point determination, and demonstration of cross-reacting enzyme from various tissue sources.     

A semi-automated assessment of cell size and shape in monolayers,with optional adjustment for the cell-cell border width-application to human corneal endothelium

Measurements of large numbers of feature sizes within defined domains (e.g. cell areas within cell-cell borders) can be a time-consuming activity, but automation that includes defining such domains has not be proven to be very reliable. Other alternatives are therefore needed, and the goal of the present studies was both to develop a semi-automated (interactive) measurement system for cell areas and to carefully compare the output to that obtained using a manual digitiser pad method. A particular interest was in the contribution made by the cell-cell border zones. Non-contact specular micrographs of central corneal endothelium were obtained from 20 white male adults, aged 40-60 years. An overlay of the endothelial image was generated manually, from which the areas of around 200 cells were measured manually with a digitiser pad and also by a computer-assisted scanning method. The pad data was typed into a spread sheet along with details of the number of cell apices (sides). The computerised analysis identified borders of the same cells on the overlay, reduced these borders to a minimum, and then assessed cell area by the pixel count along with the number of neighbouring cells (to give cell sides data). The average cell area was 393 +/- 28 and 422 +/- 29 microm(2) (mean+/-SD) by the digitiser pad and computer-based methods, respectively. The average areas for each cell type were 153, 270, 392, 519 and 685 microm(2) for 4-, 5-, 6-, 7- and 8-sided cells, respectively. Assessment of the relationship between cell area and the number of cell sides (area-side relationships) showed a highly significant and positive correlation (P<0.001; r(2)=0.865). Comparing the two methods, the average cell area was 7.5% higher in the computer scan method, and this is attributed to the fact that the contribution made by the cell borders (the para-cellular space) had been essentially eliminated. A proportional correction factor can be applied to add back the cell borders/intercellular space to the computerised output, and examples are given based on using the average data from digitiser pad for each cell type. In conclusion, a computer assisted method has been developed to simultaneously provide data on the variance in cell areas (polymegethism) and cell shape (pleomorphism) from overlays of 200 cells from human corneal endothelial images, with the cell border zone corrected to allow for a finite para-cellular space.     

APPLIED ISSUES: Size‐dependent mortality of migratory silver eels at a hydropower plant,and implications for escapement to the sea

1. The European eel population has decreased drastically during recent decades, and new EU‐legislation calls for measures to change this negative trend. This decline has been attributed to a number of factors, including habitat fragmentation by structural barriers that prevent eels moving between freshwater and the sea. The success of downstream migrating adult silver eels migrating past a hydroelectric plant (HEP) in Sweden was examined by radio‐telemetry, and the results were considered in a historical context by analysing catch data from the river for 1957–2006. 2. The choice of routes and passage success were quantified for three treatment groups and one control group of silver eels. The first treatment, the reservoir group (n = 50), was released into the reservoir upstream of the HEP, and these fish could proceed downstream by passing through the HEP (20 mm rack and turbines) or by entering the spill gates into the former channel, bypassing the HEP. The second treatment group (inside rack, n = 15) was released downstream of the 20‐mm rack and had to pass through the turbines to continue migration to the sea. The third treatment group consisted of dead radio‐tagged eels (n = 6) that were released into the turbines to study the extent of drifting by dead individuals. Finally, the control group (n = 50) was released downstream of the HEP to test for effects of confounding factors. 3. Most live individuals displayed migratory behaviour and continued to proceed downstream after release. Only 8% of the fish released in the reservoir or downstream of the HEP (control) did not migrate. The probability of reaching the next HEP, 24 km further downstream, was high for the control group (96%) and the reservoir‐released individuals that passed the HEP via the spill gates and the former channel (83%). Survival was low and size‐dependent for the individuals that passed the turbines (40%) and even lower for the individuals that had to pass through the rack and the turbines (26%). The overall passage success for eels released in the reservoir was 30%, including both routes. 4. Annual catch data from 1957 to 2006 showed that the number of eels in the River Ätran has decreased. Despite this decrease, escapement biomass has remained unchanged, because of the fact that the mean size of eels has doubled. Passage data from 2007 show that changes in size and abundance have resulted in a reduction of relative escapement to the sea to values that are 21–24% of what they were in 1957–66. However, this low level of escapement could potentially be rectified if appropriate measures facilitating HEP passage are successfully implemented, since the potential escapement biomass in the river, owing to the large size of the eels, has changed little since the 1950s.     

Assessment of methods for covalent binding of nucleic acids to magnetic beads, Dynabeads, and the characteristics of the bound nucleic acids in hybridization reactions.

Dynabeads are magnetic monosized beads with high stability, high uniformity, unique paramagnetic properties, low particle-particle interaction, and high dispersibility. Different reactive groups; hydroxyl, carboxyl and amino groups can be attached to the surface. Several methods for covalent attachment of DNA or oligonucleotides to the beads were investigated. Best coupling yields were obtained by carbodiimide-mediated end-attachment of 5'-phosphate and 5'-NH2 modified nucleic acids to respectively amino and carboxyl beads. The carboxyl beads showed a low degree of non-specific binding, while a better yield of end-attached nucleic acids was obtained using the amino beads. The DNA-beads worked efficiently in hybridization experiments, and the kinetics of hybridization approach those of solution hybridization.     

Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation,sugar hydrolysis,Maillard reactions and their relationship to glass state transition

Two primary biochemical reactions in seed ageing (lipid peroxidation and non-enzymatic protein glycosylation with reducing sugars) have been studied under different seed water contents and storage temperatures, and the role of the glassy state in retarding biochemical deterioration examined. The viability loss of Vigna radiata seeds during storage is associated with Maillard reactions; however, the contribution of primary biochemical reactions varies under different storage conditions. Biochemical deterioration and viability loss are greatly retarded in seeds stored below a high critical temperature (approximately 40 degrees C above glass transition temperature). This high critical temperature corresponds to the cross-over temperature (T(c)) of glass transition where molecular dynamics changes from a solid-like system to a normal liquid system. The data show that seed ageing slows down significantly, even before seed tissue enters into the glassy state.     

HLA-B*2704, an allotype associated with ankylosing spondylitis, is critically dependent on transporter associated with antigen processing and relatively independent of tapasin and immunoproteasome for maturation, surface expression, and T cell recognition: relationship to B*2705 and B*2706

B*2704 is strongly associated to ankylosing spondylitis in Asian populations. It differs from the main HLA-B27 allotype, B*2705, in three amino acid changes. We analyzed the influence of tapasin, TAP, and immunoproteasome induction on maturation, surface expression, and T cell allorecognition of B*2704 and compared some of these features with B*2705 and B*2706, allotypes not associated to disease. In the tapasin-deficient .220 cell line, this chaperone significantly influenced the extent of folding of B*2704 and B*2705, but not their egress from the endoplasmic reticulum. In contrast, B*2706 showed faster folding and no accumulation in the endoplasmic reticulum in the absence of tapasin. Surface expression of B*2704 was more tapasin dependent than B*2705. However, expression of free H chain decreased in the presence of this chaperone for B*2705 but not B*2704, suggesting that more suboptimal ligands were loaded on B*2705 in the absence of tapasin. Despite its influence on surface expression, tapasin had little effect on allorecognition of B*2704. Both surface expression and T cell recognition of B*2704 were critically dependent on TAP, as established with TAP-deficient and TAP-proficient T2 cells. Both immunoproteasome and surface levels of B*2704 were induced by IFN-gamma, but this had little effect on allorecognition. Thus, except for the differential effects of tapasin on surface expression, the tapasin, TAP, and immunoproteasome dependency of B*2704 for maturation, surface expression, and T cell recognition are similar to B*2705, indicating that basic immunological features are shared by the two major HLA-B27 allotypes associated to ankylosing spondylitis in human populations.     

Two mechanisms for inhibition of ADP-induced platelet shape change by 5'-p-fluorosulfonylbenzoyladenosine. Conversion to adenosine, and covalent modification at an ADP binding site distinct from that which inhibits adenylate cyclase

The interaction of ADP with platelets leads to shape change, exposure of fibrinogen binding sites, and aggregation, all of which have been shown to be inhibited by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an alkylating analogue of adenine nucleotides which binds covalently to a 100-kDa polypeptide in intact platelet membranes (Figures, W. R., Niewiarowski, S., Morinelli, T., Colman, R. F., and Colman, R. W. (1981) J. Biol. Chem. 256, 7789-7795). In plasma, FSBA can break down to adenosine which stimulates adenylate cyclase. To distinguish between direct effects of FSBA and the actions of adenosine, we have used washed platelet suspensions and adenosine deaminase. We studied the effects of FSBA on shape change and cyclic AMP metabolism, and on the binding of 2-methylthio-ADP, which mimics the effects of ADP on cyclic AMP metabolism at concentrations too low to activate platelets. Inhibition of ADP-induced shape change of platelets incubated with FSBA for 2 min in platelet-rich plasma was greatly reduced by adenosine deaminase. In the presence of a phosphodiesterase inhibitor, 100 microM FSBA increased platelet cyclic AMP to the same extent as did 10 microM adenosine. These effects were inhibited by theophylline, an adenosine receptor antagonist, and by adenosine deaminase. Incubation of washed platelets for 60 min with FSBA and adenosine deaminase caused a concentration-dependent inhibition of ADP-induced shape change. Inhibition closely paralleled the covalent incorporation of 3H from tritiated FSBA into platelet membranes. Under these conditions, FSBA did not block inhibition of cyclic AMP accumulation by ADP, nor did it block the binding of 2-methylthio-ADP. We conclude that part of the inhibition of shape change caused by brief exposure to FSBA is due to adenosine, but at longer times shape change is inhibited in association with covalent incorporation of sulfonylbenzoyladenosine. This effect of FSBA is independent of adenosine and occurs at a site distinct from that at which ADP inhibits adenylate cyclase.