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What neural mechanisms underlie the ability to attend to a complex object in the presence of competing overlapping stimuli? We evaluated whether object-based attention might involve pattern-specific feedback to early visual areas to selectively enhance the set of low-level features corresponding to the attended object. Using fMRI and multivariate pattern analysis, we found that activity patterns in early visual areas (V1-V4) are strongly biased in favor of the attended object. Activity patterns evoked by single faces and single houses reliably predicted which of the 2 overlapping stimulus types was being attended with high accuracy (80-90% correct). Superior knowledge of upright objects led to improved attentional selection in early areas. Across individual blocks, the strength of the attentional bias signal in early visual areas was highly predictive of the modulations found in high-level object areas, implying that pattern-specific attentional filtering at early sites can determine the quality of object-specific signals that reach higher level visual areas. Through computational modeling, we show how feedback of an average template to V1-like units can improve discrimination of exemplars belonging to the attended category. Our findings provide a mechanistic account of how feedback to early visual areas can contribute to the attentional selection of complex objects., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Glycans manifest in conjunction with the broad spectrum O-linked protein glycosylation in species within the genus Neisseria display intra- and interstrain diversity. Variability in glycan structure and antigenicity are attributable to differences in the content and expression status of glycan synthesis genes. Given the high degree of standing allelic polymorphisms in these genes, the level of glycan diversity may exceed that currently defined. Here, we identify unique protein-associated disaccharide glycoforms that carry N-acetylglucosamine (GlcNAc) at their non-reducing end. This altered structure was correlated with allelic variants of pglH whose product was previously demonstrated to be responsible for the expression of glucose (Glc)-containing disaccharides. Allele comparisons and site-specific mutagenesis showed that the presence of a single residue, alanine at position 303 in place of a glutamine, was sufficient for GlcNAc versus Glc incorporation. Phylogenetic analyses revealed that GlcNAc-containing disaccharides may be widely distributed within the pgl systems of Neisseria particularly in strains of N. meningitidis. Although analogous minimal structural alterations in glycosyltransferases have been documented in association with lipopolysaccharide and capsular polysaccharide variability, this appears to be the first example in which such changes have been implicated in glycan diversification within a bacterial protein glycosylation system., (© 2014 John Wiley & Sons Ltd.)

Symmetry is a biologically relevant, mathematically involving, and aesthetically compelling visual phenomenon. Mirror symmetry detection is considered particularly rapid and efficient, based on experiments with random noise. Symmetry detection in natural settings, however, is often accomplished against structured backgrounds. To measure salience of symmetry in diverse contexts, we assembled mirror symmetric patterns from 101 natural textures. Temporal thresholds for detecting the symmetry axis ranged from 28 to 568 ms indicating a wide range of salience (1/Threshold). We built a model for estimating symmetry-energy by connecting pairs of mirror-symmetric filters that simulated cortical receptive fields. The model easily identified the axis of symmetry for all patterns. However, symmetry-energy quantified at this axis correlated weakly with salience. To examine context effects on symmetry detection, we used the same model to estimate approximate symmetry resulting from the underlying texture throughout the image. Magnitudes of approximate symmetry at flanking and orthogonal axes showed strong negative correlations with salience, revealing context interference with symmetry detection. A regression model that included the context-based measures explained the salience results, and revealed why perceptual symmetry can differ from mathematical characterizations. Using natural patterns thus produces new insights into symmetry perception and its possible neural circuits.

Most moving objects in the world are non-rigid, changing shape as they move. To disentangle shape changes from movements, computational models either fit shapes to combinations of basis shapes or motion trajectories to combinations of oscillations but are biologically unfeasible in their input requirements. Recent neural models parse shapes into stored examples, which are unlikely to exist for general shapes. We propose that extracting shape attributes, e.g., symmetry, facilitates veridical perception of non-rigid motion. In a new method, identical dots were moved in and out along invisible spokes, to simulate the rotation of dynamically and randomly distorting shapes. Discrimination of rotation direction measured as a function of non-rigidity was 90% as efficient as the optimal Bayesian rotation decoder and ruled out models based on combining the strongest local motions. Remarkably, for non-rigid symmetric shapes, observers outperformed the Bayesian model when perceived rotation could correspond only to rotation of global symmetry, i.e., when tracking of shape contours or local features was uninformative. That extracted symmetry can drive perceived motion suggests that shape attributes may provide links across the dorsal-ventral separation between motion and shape processing. Consequently, the perception of non-rigid object motion could be based on representations that highlight global shape attributes.

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR-directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1-104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (K(D) value of 49 and 10 pmol/L, respectively) compared with IGF-I (approximately 10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor-binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 A. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I-induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A.

Elevated expression of insulin-like growth factor II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon and liver cancer. As IGF-II can deliver a mitogenic signal through both the type 1 insulin-like growth factor receptor (IGF-IR) and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is an attractive therapeutic option. One method of doing this would be to find antibodies that bind tightly and specifically to the peptide, which could be used as protein therapeutics to lower the peptide levels in vivo and/or to block the peptide from binding to the IGF-IR or IR-A. To address this, Fabs were selected from a phage-display library using a biotinylated precursor form of the growth factor known as IGF-IIE as a target. Fabs were isolated that were specific for the E-domain C-terminal extension and for mature IGF-II. Four Fabs selected from the library were produced, complexed with IGF-II and set up in crystallization trials. One of the Fab-IGF-II complexes (M64-F02-IGF-II) crystallized readily, yielding crystals that diffracted to 2.2 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.7, b = 106.9, c = 110.7 A. There was one molecule of the complete complex in the asymmetric unit. The same Fab was also crystallized with a longer form of the growth factor, IGF-IIE. This complex crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 50.7, b = 107, c = 111.5 A, and also diffracted X-rays to 2.2 A resolution.

Aims: The primary aim was to compare the efficacy of smoking cessation treatment using a combination of active nicotine patch plus active nicotine gum versus therapy consisting of active nicotine patch plus placebo gum in a sample of alcohol-dependent tobacco smokers in an early phase of out-patient alcohol treatment. A secondary aim was to determine whether or not there were any carry-over effects of combination nicotine replacement on drinking outcomes., Design: Small-scale randomized double-blind placebo-controlled clinical trial with 1-year smoking and drinking outcome assessment., Setting: Two out-patient substance abuse clinics provided a treatment platform of behavioral alcohol and smoking treatment delivered in 3 months of weekly sessions followed by three monthly booster sessions., Participants: Participants were 96 men and women with a diagnosis of alcohol abuse or dependence and smoking 15 or more cigarettes per day., Intervention: All participants received open-label transdermal nicotine patches and were randomized to receive either 2 mg nicotine gum or placebo gum under double-blind conditions., Findings: Analysis of 1-year follow-up data revealed that patients receiving nicotine patch plus active gum had better smoking outcomes than those receiving patch plus placebo gum on measures of time to smoking relapse and prolonged abstinence at 12 months. Alcohol outcomes were not significantly different across medication conditions., Conclusions: Results of this study were consistent with results of larger trials of smokers without alcohol problems, showing that combination therapy (nicotine patch plus gum) is more effective than monotherapy (nicotine patch) for smoking cessation.

We investigated perceptual segmentation in the context of a perceived-orientation task. Stimuli were dot clusters formed by the union of a large elliptical sub-cluster and a secondary circular sub-cluster. We manipulated the separation between the two sub-clusters, their common dot density, and the size of the secondary sub-cluster. As the separation between sub-clusters increased, the orientation perceived by observers shifted gradually from the global principal axis of the entire cluster to that of the main sub-cluster alone. Thus, with increasing separation, the dots within the secondary sub-cluster were assigned systematically lower weights in the principal-axis computation. In addition, this shift occurred at smaller separations for higher dot densities-consistent with the idea that reliable segmentation is possible with smaller separations when the dot density is high. We propose that the visual system employs a robust statistical estimator in this task and that data points are weighted differentially based on the likelihood that they arose from a separate generative process. However, unlike in standard robust estimation, weights based on residuals are insufficient to characterize human segmentation. Rather, these must be computed based on more comprehensive generative models of dot clusters.

Contrary to the conventional assumption that humans perceive shapes of rigid objects as constant despite retinal-image variations caused by changes in orientation and position, we show that the depths of three-dimensional (3-D) textured shapes appear to vary when the image is rotated. In paired comparisons of static stimuli, depth amplitude was perceived to be greater at vertical than at oblique orientations. A similar oblique bias was found for simple two-dimensional (2-D) obtuse angles. Using projective geometry to link angle magnitude to the orientation flows that convey 3-D shape from texture cues, we show quantitatively that the 2-D bias predicts the 3-D bias. Our finding that perception of angular magnitude is dependent on orientation has broad implications for shape constancy because orientation flows have also been implicated in 3-D perception from reflections and shading, and contour curvature is fundamental to uncovering depth and part-structure of shapes. We examined the roles played in the observed biases by anisotropies in numbers and tuning widths of orientation-tuned cells in striate cortex as well as the distribution of oriented energy in natural scenes. An optimal stimulus decoding model for 2-D angles revealed that the narrower tuning of cells for horizontal orientations combined with cross-orientation inhibition explains the orientation-dependent angle distortion and hence the 3-D shape inconstancy. Variations in properties within neural populations can thus have direct effects on visual perceptions and need to be included in neural decoding models.

The geometric determinants of shape decomposition were studied using a performance-based method. Observers' identification of contour segments was shown to be systematically modulated by their curvature properties, and by the geometric properties of the enclosed region. Specifically, negative minima of contour curvature provided the best segment boundaries. Segments with negative-minima boundaries were identified with greater accuracy than those with positive maxima or inflection boundaries of comparable length. Additionally, segment identification was shown to be determined by contour length, the turning angle at part boundaries, and the width at the part's base (hence the part's protrusion). The results indicate that part decomposition is an automatic process. Moreover, this process is graded, i.e. parts are more strongly segmented, or more likely to be perceived, according to the strength of many geometric determinants.

Saccades aimed at spatially extended targets land reliably at central locations determined by pooling information across the target shape [Melcher, D., & Kowler, E. (1999). Shape, surfaces and saccades. Vision Research, 39, 2929-2946; Vishwanath, D., & Kowler, E. (2003). Localization of shapes: Eye movements and perception compared. Vision Research, 43, 1637-1653]. Previous findings of saccadic errors when attempting to look at a target in the midst of distractors encouraged suggestions that pooling occurs indiscriminately, with little or no influence of a selective filter to eliminate the influence of nearby distractors. To determine the effectiveness of filtering, saccadic localization was studied for saccades made to a set of target elements (discs) interleaved with an equivalent set of distractors of a different color. With such interleaved elements, selection and spatial pooling are constrained to occur over the same spatial region. The results showed that filtering was effective and saccadic landing position was determined mainly by the target elements. Concurrent perceptual judgments made about the same stimuli (estimating the mean size of either target or distractor discs) showed better performance for the target discs than distractors, confirming that perceptual attention was allocated to the set of target elements. These results: (1) support the role of attention in setting the input to the spatial pooling process that guides saccades to spatially extended targets, and (2) show that perceptual judgments of mean value, often thought to impose modest attentional demands, are not immune to the constraints of this pre-saccadic filter.

The classical oblique effect refers to the finding that observers are faster and more accurate in discriminating the orientation of a line or edge when it is at or near vertical or horizontal than when it is at an oblique orientation (Appelle, 1972; Mach, 1861). Based on the finding that observer sensitivity to orientation of simple symmetric shapes like an ellipse or the letter "X" also exhibits an oblique effect, Li and Westheimer (1997) suggested that the effect does not arise solely from inequality of simple orientation-tuned receptors in early visual processing, but also involves later orientation processing that can encompass more complex inputs such as shape axes. In this work, we examined how the oblique effect impacts three-dimensional shapes defined by texture cues.

LFA-1 (alpha(L)beta(2)) mediates cell-cell and cell-extracellular matrix adhesions essential for immune and inflammatory responses. One critical mechanism regulating LFA-1 activity is the conformational change of the ligand-binding alpha(L) I domain from low-affinity (LA), closed form, to the high-affinity (HA), open form. Most known integrin antagonists bind both forms. Antagonists specific for the HA alpha(L) I domain have not been described. Here, we report the identification and characterization of a human antibody AL-57, which binds to the alpha(L) I domain in a HA but not LA conformation. AL-57 was discovered by selection from a human Fab-displaying library using a locked-open HA I domain as target. AL-57 Fab-phage bound HA I domain-expressing K562 cells (HA cells) in a Mg(2+)-dependent manner. AL-57 IgG also bound HA cells and PBMCs, activated by Mg(2+)/EGTA, PMA, or DTT. The binding profile of AL-57 IgG on PBMCs was the same as that of ICAM-1, the main ligand of LFA-1. In contrast, an anti-alpha(L) murine mAb MHM24 did not distinguish between the HA and LA forms. Moreover, AL-57 IgG blocked ICAM-1 binding to HA cells with a potency greater than MHM24. It also inhibited ICAM-1 binding to PBMCs, blocked adhesion of HA cells to keratinocytes, and inhibited PHA-induced lymphocyte proliferation with potencies comparable with MHM24. These results indicate that specifically targeting the HA I domain is sufficient to inhibit LFA-1-mediated, adhesive functions. AL-57 represents a therapeutic candidate for treatment of inflammatory and autoimmune diseases.

Affinity of integrin lymphocyte function-associated antigen 1 (LFA-1) is enhanced by conformational changes from the low-affinity closed form to the high-affinity (HA) open form of the ligand-binding inserted (I) domain as shown by work with purified I domains. However, affinity up-regulation of LFA-1 on the cell surface by physiological agonists such as chemokines has yet to be demonstrated by monovalent reagents. We characterize a mAb, AL-57 (activated LFA-1 clone 57), that has been developed by phage display that selectively targets the HA open conformation of the LFA-1 I domain. AL-57 discriminates among low-affinity, intermediate-affinity, and HA states of LFA-1. Furthermore, AL-57 functions as a ligand mimetic that binds only upon activation and requires Mg2+ for binding. Compared with the natural ligand intercellular adhesion molecule-1, AL-57 shows a tighter binding to the open I domain and a 250-fold slower off rate. Monovalent Fab AL-57 demonstrates affinity increases on a subset (approximately 10%) of lymphocyte cell surface LFA-1 molecules upon stimulation with CXCL-12 (CXC chemokine ligand 12). Affinity up-regulation correlates with global conformational changes of LFA-1 to the extended form. Affinity increase stimulated by CXCL-12 is transient and peaks 2 to 5 min after stimulation.

Although the orientation of line segments and simple shapes is a well-studied area of vision, little is known about geometric factors that influence perceived orientation of complex multipart shapes. The study of these factors is of interest because it allows for an insight into the basic problem of how local geometric attributes are integrated perceptually into a global shape representation. We examined the perceived orientation of two-part shapes using an adjustment method and a 2AFC task. In particular, we investigated the influence of the perceptual salience, or distinctiveness, of a part--as defined by the turning angles at its boundaries--and its area relative to the main "base" part. In contrast to previous results on simple shapes, our results exhibited large and systematic deviations of perceived orientation from the principal axis of the shape. For shapes with sharp part boundaries, perceived global orientation deviated maximally from the principal axis and was approximated by the axis of the main base part of the shape. With weakening part boundaries, the perceived orientation gradually approached the principal axis of the entire shape, reflecting that both parts were taken into account in estimating orientation. The results are consistent with a differentially weighted principal-axis computation in which the attached part is given systematically lower weighting with increasing turning angles at the part boundaries. They thus allow a quantitative characterization of part salience in terms of part independence: Turning angles at a part's boundaries determine the extent to which its influence is perceptually separable from the rest of the shape. We suggest that Robust Statistics may provide a useful framework for quantifying the influence of part segmentation on visual estimation.

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

Many recent findings suggest that human observers are surprisingly "blind" to changes in visual displays, failing to notice when substantial scene elements are added, subtracted, or altered in successive presentations of the scene. But observers are far more sensitive to certain visual changes than others, and we suggest that which types of changes enjoy differential sensitivity can reveal a great deal about the underlying visual representations. In this study, we investigate how the human visual system represents the shape of objects by demonstrating a previously unknown influence on detection of changes in shape: the sign of contour curvature. We show that subjects are substantially more sensitive to changes in concave regions of a shape's contour than to changes in convex regions, even when these changes do not alter the number or location of parts. Further, we show that this effect is modulated by figure-ground assignment, so that changes to the same physical contour are more or less detectable, depending on the contour's perceived figural status, which determines whether the change falls in a concave or convex region. The results demonstrate a heightened sensitivity for changes at concavities that is not reducible to a sensitivity to changes in gross part structure.

Shape representation was studied using a change detection task. Observers viewed two individual shapes in succession, either identical or one a slightly altered version of the other, and reported whether they detected a change. We found a dramatic advantage for concave compared to convex changes of equal magnitude. Observers were more accurate when a concavity along the contour was introduced, or removed, compared to a convexity. This result sheds light on the underlying representation of visual shape, and in particular the central role played by part-boundaries. Moreover, this finding shows how change detection methodology can serve as a useful tool in studying the specific form of visual representations.

Several phage isolates that bind specifically to human serum albumin (HSA) were isolated from disulfide-constrained cyclic peptide phage-display libraries. The majority of corresponding synthetic peptides bind with micromolar affinity to HSA in low salt at pH 6.2, as determined by fluorescence anisotropy. One of the highest affinity peptides, DX-236, also bound well to several mammalian serum albumins (SA). Immobilized DX-236 quantitatively captures HSA from human serum; mild conditions (100 mM Tris, pH 9.1) allow release of HSA. The DX-236 affinity column bound HSA from human serum with a greater specificity than does Cibacron Blue agarose beads. In addition to its likely utility in HSA and other mammalian SA purifications, this peptide media may be useful in the proteomics and medical research markets for selective removal of mammalian albumin from serum prior to mass spectrometric and other analyses.

The purpose of this study was to find out the functional and anatomical relationship between the various cardiac segments in congenital heart disease before and after birth. This study of cardiac pathology focused on 227 fetuses referred to fetal echocardiography because of suspicion of congenital heart defects (CHD) from 1986 to 1991. The heart was imaged and classified according to a division into 7 adjacent segments. Normality or abnormality of each segment and of the general state of the subject's heart was coded in a binary mode, in which 0 = normal and 1 = pathological. We also coded the extracardiac morphology, noting existence or absence of another extracardiac defect in the fetus. The binary segmental data were entered into a worksheet for further analysis. Observations were conducted prenatally and verified after birth or abortion. Fetal echocardiography was first assessed using conventional statistical analysis. The calculated accuracy and predictive values to diagnose any cardiac defect as well as for correctly coding abnormal or normal cardiac segments were excellent. Then, using HUDAP software (Hebrew University Data Analysis Package), we could demonstrate a perfect fit between the functional and pathological structure of the cardiac segments in the fetus. Moreover, using this unique type of data analysis, we found a multidimensional scaling confirmation of the embryogenesis theory of the heart., (Copyright 2000 S. Karger AG, Basel.)

The seed mucilage from Plantago major L. contains acidic heteroxylan polysaccharides. For further structural analysis, oligosaccharides were generated by partial acid hydrolysis and then isolated by high-pH anion-exchange chromatography (HPAEC). Each HPAEC fraction was shown by ESMS to contain one major oligosaccharide and several minor components. Partial structures of the oligosaccharides were determined using GC-MS, ESMS and ES tandem mass spectrometry (ESMS/MS). A (1-->4)-linked xylan trisaccharide and (1-->3)-linked xylan oligosaccharides with DP 6-11 suggested that the backbone of the heteroxylan polysaccharide consisted of blocks of (1-->4)-linked and (1-->3)-linked Xylp residues. A (1-->2)-linked Xylp disaccharide and a branched tetrasaccharide were also found, revealing that single Xylp residues are linked to the O-2 of some of the (1-->4)-linked Xylp residues in the backbone. In addition, our results confirm the presence of side chains consisting of the disaccharide GlcpA-(1-->3)-Araf.

The 9804 gene, which encodes a human Ly-6 protein most similar to mouse differentiation Ag TSA-1/Sca-2, has also been called RIG-E. Like mouse TSA-1, it has a broad tissue distribution with varied expression levels in normal human tissues and tumor cell lines. Like some members of the murine Ly-6 family, the 9804 gene is responsive to IFNs, particularly IFN-alpha. Overlapping genomic fragments spanning the 9804 gene (5543 bp) have been isolated and characterized. The gene organization is analogous to that of known mouse Ly-6 genes. The first exon, 2296 bp upstream from exon II, is entirely untranslated. The three coding exons (II, III, and IV) are separated by short introns of 321 and 131 bp, respectively. Primers were developed for specific amplification of 9804 gene fragments. Screening of human-hamster somatic cell hybrids and yeast artificial chromosomes (YACs) indicated that the gene is distal to c-Myc, located in the q arm of human chromosome 8. No positives were detected from the Centre d'Etude du Polymorphisme Humain mega-YAC A or B panels, nor from bacterial artificial chromosome libraries; two positive cosmids (c101F1 and c157F6) were isolated from a human chromosome 8 cosmid library (LA08NC01). Fluorescence in situ hybridization of metaphase spreads of chromosome 8, containing hybrid cell line 706-B6 clone 17 (CL-17) with cosmid c101F1, placed the 9804 gene close to the telomere at 8q24.3. This mapping is significant, since the region shares a homology with a portion of mouse chromosome 15, which extends into band E where Ly-6 genes reside. Moreover, the gene encoding E48, the homologue of mouse Ly-6 molecule ThB, has also been mapped to 8q24.

Aspergillus fumigatus grown in submerged and surface cultures was extracted, and the extracts were analyzed separately. The submerged extract contained 31.9% protein and 8.3% carbohydrate, while the corresponding values were 17.0% and 33.3% for the surface material. With individual sera from patients with allergic asthma, SDS-PAGE combined with immunoblotting revealed that the submerged extract contained at least six strong IgE-binding components (20, 30, 38, 50, 68, and 90 kDa) in addition to several weak to medium IgE-binding components. The surface extract contained about the same number of IgE-binding components, but only one gave a strong reaction (20 kDa). The allergens present were shown to have pI between 4.5 and 5.6 as demonstrated by isoelectric focusing (IEF) combined with immunoblotting. For identification of A. fumigatus glycoprotein allergens, both extracts were treated with periodate under mild conditions. Two allergens of the submerged extract (90 and 38 kDa) partly lost their IgE-binding ability by this treatment, indicating that these components are glycoproteins and that the carbohydrate moiety is involved in the IgE binding. The IgE-binding ability of the 20-kDa allergen was not influenced by periodate. For assessment of the stability of the two allergen extracts, aqueous solutions were kept at 4 degrees C for 2, 7, and 21 d and then analyzed by SDS-PAGE and immunoblotting. The results showed that most allergens of the submerged extract were partly inactivated after 2 d. After 21 d, only the 20-kDa and 30-kDa components were still able to bind IgE. Similar results were obtained by analyzing the surface extract.(ABSTRACT TRUNCATED AT 250 WORDS)

Human gamma-glutamyl transpeptidase (GGT)1 is composed of two subunits derived from a single precursor (Nash, B., and Tate, S.S. (1984) J. Biol. Chem. 259, 678-685; Finidori, J., Laperche, Y., Tsapis, R., Barouki, R., Guellaën, G., and Hanoune, J. (1984) J. Biol. Chem. 259, 4687-4690) consisting of 569 amino acids (Laperche, Y., Bulle, F., Aissani, T., Chobert, M.N., Aggerbeck, M., Hanoune, J., and Guellaën, G. (1986) Proc Natl. Acad. Sci. U.S.A. 83, 937-941). In the present study we report the cloning of an altered form of this precursor from human liver. We have isolated two clones, one 2,632 base pairs (bp) long from a fetal liver cDNA library and one 926 bp long from an adult liver cDNA library, each containing a 22-bp insertion that introduces a premature stop codon and shortens the open reading frame to 1,098 bp when compared with known human cDNA sequences specific for GGT. Sequence analysis of a human genomic GGT clone shows that this insertion of 22 bp is generated by a splicing event involving an alternative 3'-acceptor site. By polymerase chain reaction experiments we demonstrate that the alternatively spliced mRNA is present in polysomes from the microsomal fraction of a human hepatoma cell line (Hep G2) and thus could encode an altered GGT molecule of 39,300 Da (366 amino acids) encompassing most of the heavy subunit which is normally 41,500 Da (380 amino acids). The altered mRNA is detected in various human tissues including liver, kidney, brain, intestine, stomach, placenta, and mammary gland. This report is the first demonstration of an alternative primary sequence in the mRNA coding for GGT, a finding that could be related to the presence of some inactive forms of GGT detected in human tissues.

Transient acantholytic dermatosis is a self-limiting benign disease. It is characterized by multiple pruritic erythematous papules and papulovesicles found predominantly on the trunk and extremities. This primary acantholytic dermatosis affects individuals older than 40 years. We present a case study of an individual who received a regimen of isotretinoin (Accutane) for treatment of severe pruritus after conventional forms of therapy failed to alleviate his condition and abate the formation of new lesions.

We have investigated transcription termination on a segment of Drosophila DNA that complements a yeast adenine-8 mutation. Poly(A)+ RNA transcribed from this segment in yeast terminates at multiple sites clustered just beyond an AAUAAA sequence implicated in polyadenylation of higher eucaryotic messages. Deletion analysis indicates that, in yeast, this sequence is not required for polyadenylation. Rather, transcription termination is signalled by a region that is upstream of the AAUAAA sequence. At least part of the control region appears to be an 8-base pair (bp) sequence also found in the termination control region of the yeast CYC1 gene. Termination sites for the various deletions show a clear sequence preference. These sites occur in clusters at least 50 bp downstream of the control region, suggesting similarities between termination in yeast and p-dependent termination in bacteria.

We have mapped a signal sequence for mRNA 3'-end formation in Saccharomyces cerevisiae by using a Drosophila melanogaster DNA segment that complements a yeast adenine-8 mutation. That the 3' end of the transcript in S. cerevisiae nearly coincides with that in D. melanogaster is consistent with the possibility that mRNA termini are similarly determined in both organisms. Deletion analysis reveals that the complete signal is no more than 21 base pairs long. Part of the signal is the sequence TTTTTATA, which is seen in the termination region of several yeast genes. TTTTTATA appears to be able to act autonomously as a partial termination signal. The efficiency of the complete signal is affected by substitution of sequences downstream from it. This modulation of the effect of a signal is consistent with termination in S. cerevisiae, resembling rho-dependent termination in bacteria.

In vitro synthesized RNAs complementary to the three satellite DNAs of Drosophila virilis have been used in a series of in situ hybridization experiments with polytene chromosomes from virilis group species. Gall and Atherton (1974) demonstrated that each of the satellites of D. virilis is comprised of many repeats of a distinct, seven base pair long, simple sequence. With few exceptions, copies of each of these simple sequences are detected in the chromocenters of all virilis group species. This is true even in species which do not possess satellite DNAs at buoyant densities corresponding to those of the satellite DNAs of D. virilis. Small quantities of the three simple sequences are also detected in euchromatic arms of several different species. The same euchromatic location may contain detectable copies of one, two, or all three simple sequence DNAs. The amounts of simple sequences at each location in the euchromatin may vary between species, between different stocks of the same species, and even between individuals of the same stock. The simple sequences located in the euchromatin appear to undergo DNA replication during formation of polytene chromosomes unlike those in heterochromatin. The locations of the euchromatic sequences are not the results of single chromosomal inversion events involving heterochromatic and euchromatic breakpoints.

The DNAs from two virilis group species of Drosophila, D. lummei and D. kanekoi, have been analyzed. D. lummei DNA has a major satellite which, on the basis of CsCl equilibrium centrifugation, thermal denaturation, renaturation and in situ hybridization is identical to D. virilis satellite I. D. kanekoi DNA has a major satellite at the same buoyant density in neutral CsCl gradients as satellite III of D. virilis. However, on the basis of alkaline CsCl gradients, the satellite contains a major and a minor component, neither one of which is identical to D. virilis satellite III. By in situ hybridization experiments, sequences complementary to the major component of the D. kanekoi satellite are detected in only some species and in a way not consistent with the phylogeny of the group. However, by filter hybridization experiments using nick-translated D. kanekoi satellite as well as D. lummei satellite I and D. virilis satellite III DNAs as probes, homologous sequences are detected in the DNAs of all virilis group species. Surprisingly, sequences homologous to these satellite DNAs are detected in DNAs from non-virilis group Drosophila species as well as from yeast, sea urchin, Xenopus and mouse.