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Crystal structure of the interleukin-4/receptor alpha chain complex reveals a mosaic binding interface.

Interleukin-4 (IL-4) is a principal regulatory cytokine during an immune response and a crucial determinant for allergy and asthma. IL-4 binds with high affinity and specificity to the ectodomain of the IL-4 receptor alpha chain (IL4-BP). Subsequently, this intermediate complex recruits the common gamma chain (gamma c), thereby initiating transmembrane signaling. The crystal structure of the intermediate complex between human IL-4 and IL4-BP was determined at 2.3 A resolution. It reveals a novel spatial orientation of the two proteins, a small but unexpected conformational change in the receptor-bound IL-4, and an interface with three separate clusters of trans-interacting residues. Novel insights on ligand binding in the cytokine receptor family and a paradigm for receptors of IL-2, IL-7, IL-9, and IL-15, which all utilize gamma c, are provided.

Amino Acid Sequence↗

Sticky business: orchestrating cellular signals at adherens junctions.

Cohesive sheets of epithelial cells are a fundamental feature of multicellular organisms and are largely a product of the varied functions of adherens junctions. These junctions and their cytoskeletal associations contribute heavily to the distinct shapes, polarity, spatially oriented mitotic spindle planes, and cellular movements of developing tissues. Deciphering the underlying mechanisms that govern these conserved cellular rearrangements is a prerequisite to understanding vertebrate morphogenesis.

Actins↗

Self-motion perception: assessment by computer-generated animations.

The goal of this research is more precise description of adaptation to sensory rearrangements, including microgravity, by development of improved procedures for assessing spatial orientation perception. Thirty-six subjects reported perceived self-motion following exposure to complex inertial-visual motion. Twelve subjects were assigned to each of 3 perceptual reporting procedures: (a) animation movie selection, (b) written report selection and (c) verbal report generation. The question addressed was: do reports produced by these procedures differ with respect to complexity and reliability? Following repeated (within-day and across-day) exposures to 4 different "motion profiles," subjects either (a) selected movies presented on a laptop computer, or (b) selected written descriptions from a booklet, or (c) generated self-motion verbal descriptions that corresponded most closely with their motion experience. One "complexity" and 2 reliability "scores" were calculated. Contrary to expectations, reliability and complexity scores were essentially equivalent for the animation movie selection and written report selection procedures. Verbal report generation subjects exhibited less complexity than did subjects in the other conditions and their reports were often ambiguous. The results suggest that, when selecting from carefully written descriptions and following appropriate training, people may be better able to describe their self-motion experience with words than is usually believed.

Aerospace Medicine↗

In vivo modulation of host response and macrophage behavior by polymer networks grafted with fibronectin-derived biomimetic oligopeptides: the role of RGD and PHSRN domains.

Polymorphonuclear leukocytes, monocytes/macrophages, foreign body giant cells (FBGC), and lymphocytes are central in directing the host foreign body and inflammatory/immune reactions that impact material biostability, biocompatibility, and device efficacy. We employed the functional architecture of fibronectin to probe the mechanisms of protein-cell interaction in modulating leukocyte behavior. We demonstrated previously that the RGD and PHSRN domain of fibronectin and the spatial orientation between the motifs were crucial in regulating macrophage function in vitro. The current study delineates the role of RGD and PHSRN in modulating the host inflammatory response and macrophage behavior in vivo. Oligopeptides containing RGD and/or PHSRN domains were grafted onto a polyethyleneglycol-based substrate and subcutaneously cage implanted into rats. Peptide identity played a minimal role in modulating the host inflammatory response and adherent macrophage density. The RGD motif, either alone or at the terminal position with the PHSRN-containing flanking sequence, elicited a rapid macrophage fusion to form FBGCs at the early stage of implantation. Both the RGD motif and the PHSRN motif were important in mediating FBGC formation at the later implantation time. However, the PHSRN motif, specifically in the configuration of G3 RGDG6 PHSRNG, evoked a larger extent of FBGC coverage. Our results indicate the importance of RGD and PHSRN in modulating macrophage function in a time and orientation dependent fashion in vivo.

Amino Acid Sequence↗

Fluorescently labeled mesenchymal stem cells (MSCs) maintain multilineage potential and can be detected following implantation into articular cartilage defects.

Several studies have reported enhanced repair of damaged cartilage following implantation of mesenchymal stem cells (MSCs) into full-thickness cartilage defects suggesting that the cells in the repair tissue were derived from the implant. However, it cannot be excluded that the enhanced tissue repair is derived from host cells recruited to the defect in response to the implant, rather than the re-population of the tissue by the implanted MSCs. Our objective was to study the short-term fate of fluorescently labeled MSCs after implantation into full-thickness cartilage defects in vivo. The fluorescent dye used in our studies did not affect MSC viability or their ability to undergo osteogenic and chondrogenic differentiation in vitro. MSC gelatin constructs were implanted into full-thickness cartilage defects in goats. These cells retained the dye and were detectable by histology and flow cytometry. At intervals spanning 2 weeks post-implantation we observed gradual loss of implanted cells in the defect as well as fragments of gelatin sponge containing labeled MSCs in deep marrow spaces indicating fragmentation, dislodgement and passive migration. Fluorescent labeling enabled us to determine whether the implanted cells were lost during early time points after implantation as well as their spatial orientation throughout the defect. By determining the fate of implanted cells, new biomaterials could be engineered to correct undesirable characteristics. Testing of new biomaterials in short-term in vivo models would provide faster optimization for cell retention needed for successful, long-term cartilage regeneration.

Animals↗

Influence of different crosslinking treatments on the physical properties of collagen membranes.

The physical properties of collagen-based biomaterials are profoundly influenced by the method and extent of crosslinking. In this study, the influence of various crosslinking treatments on the physical properties of reconstituted collagen membranes was assessed. Five crosslinking agents viz., GTA, DMS, DTBP, a combination of DMS and GTA and acyl azide method were used to stabilize collagen matrices. Crosslinking density, swelling ratio, thermo-mechanical properties, stress-strain characteristics and resistance to collagenase digestion were determined to evaluate the physical properties of crosslinked matrices. GTA treatment induced the maximum number of crosslinks (13) while DMS treatment induced the minimum (7). Of the two diimidoesters (DMS and DTBP), DTBP was a more effective crosslinking agent due to the presence of disulphide bonds in the DTBP crosslinks. T(s) for DTBP and DMS crosslinked collagen were 80 degrees C and 70 degrees C, and their HIT values were 5.4 and 2.85MN/m(2), respectively. Low concentration of GTA (0.01%) increased the crosslinking density of an already crosslinked matrix (DMS treated matrix) from 7 to 12. Lowest fracture energy was observed for the acyl azide treated matrix (0.61MJ/m(3)) while the highest was observed for the GTA treated matrix (1.97MJ/m(3)). The tensile strength of GTA treated matrix was maximum (12.4MPa) and that of acyl azide treated matrix was minimum (7.2MPa). GTA, DTBP and acyl azide treated matrices were equally resistant to collagenase degradation with approximately 6% solubilization after 5h while the DMS treated was least stable with 52.4% solubilization after the same time period. The spatial orientation of amino acid side chain residues on collagen plays an important role in determining the crosslinking density and consequent physical properties of the collagen matrix.

Biocompatible Materials↗

A review of the evidence for a disengage deficit following parietal lobe damage.

We review the literature on response times to ipsilesional and contralesional targets following spatial precues in patients with damage involving the left- and right-parietal lobes with the aim of appraising the 'disengage deficit' reported initially by Posner and colleagues (Posner MI, Cohen A, Rafal RD. Neural systems control of spatial orienting. Proceedings of the Royal Society of London, B 1982;298:187-98). The data of individual subjects from a sub-sample of studies were submitted to analyses of variance, and data from all studies meeting our selection criteria were submitted to meta-analytic procedures (Hunter JE, Schmidt FL. Methods of meta-analysis: correcting error and bias in research. Newberg Park: Sagge Publications, 1990). Findings from both types of analysis conducted on data from patients with right-hemisphere lesions indicate that: (1) the disengage deficit phenomenon is robust following peripheral cues, but not following central cues; (2) the disengage deficit is large at shorter cue-target stimulus onset asynchronies (SOAs), and decreases as SOA increases; (3) the disengage deficit is larger in patients with a diagnosis of hemispatial neglect; and (4) although the magnitude of the disengage deficit appears to increase with increases in lesion size, multilobar vs unilobar involvement did not significantly alter the pattern of the disengage deficit. We also show that responses to validly cued targets in the contralesional hemispace were significantly slower than for validly cued targets in ipsilesional hemispace. Similar, but usually smaller, effects were observed in patients with homologous left-hemisphere damage. The implications of these results for current models of the role of the parietal lobes in attentional orienting are discussed.

Animals↗

Late natural history of corneal topography after excimer laser photorefractive keratectomy.

PURPOSE: To investigate the 3 month to 1 year natural history of corneal topography after excimer laser photorefractive keratectomy (PRK). DESIGN: A prospective, multicenter, nonrandomized comparative study. PARTICIPANTS: A total of 75 eyes of 68 patients with myopia were studied. INTERVENTION: Excimer laser photorefractive keratectomy and computer-assisted videokeratography were performed. MAIN OUTCOME MEASURES: Preoperative and 3, 6, and 12 month postoperative topography patterns were compared and changes assessed by averaging defined sectors of the ablation zone in individual maps to produce composite "average" topography maps. RESULTS: Corneal topography was relatively smooth 3 months after PRK. By 12 months, the corneal contour in general had become even more uniform. No "central island" effect was observed. When looking at right and left eyes independently, there was a tendency toward maximum flattening nasally. CONCLUSIONS: Corneal topography in general continues to smooth from 3 to 12 months after PRK, possibly as a result of epithelial and stromal healing and remodeling. Right and left eyes on average show mirror-image, spatially oriented topography patterns.

Adult↗

Vestibulo-oculomotor research and measurement technology for the space station era.

Recent improvements in measurement techniques and mathematical representations for eye, head and body movement have enhanced our appreciation of the complexity of spatial orientation and locomotion in three-dimensional space. The shortcomings of present measurement techniques, and their solution with emerging technologies are described. The prolonged microgravity conditions on the space station provide a unique opportunity to investigate these three-dimensional aspects of the vestibular and oculomotor systems, and in particular, the role of the otolith afferences. While the canal-ocular responses and their central pathways are reasonably well understood, the community has only recently become aware of the variety of functions fulfilled by otolith-mediated information, i.e., translational otolith-ocular reflex, inertial processing, gravitational reference, vergence control. Recent results, largely from experiments performed on the Mir Station, where the emphasis was on the otolith contribution to the vestibulo-ocular response mechanisms, are reviewed.

Humans↗

Pseudo-immunolabelling with the avidin-biotin-peroxidase complex (ABC) due to the presence of endogenous biotin in retinal Müller cells of goldfish and salamander.

Immunodetection techniques are dependent on enzyme-protein conjugates for the visualisation of antigen-antibody complexes. One of the most widely used is the avidin-biotin-peroxidase complex (ABC) method. The present study demonstrates that direct treatment of goldfish and salamander retinal sections with ABC, followed by an incubation with the chromogenic substrate 3,3-diaminobenzidine tetrahydrochloride (DAB) and H2O2, manifested a punctate staining pattern across the neural retinae, presumably through binding of avidin to endogenous biotin. Incubation with a primary antiserum against biotin followed by immunoprocessing with the peroxidase--anti-peroxidase (PAP) method showed a pattern similar to the punctuate framework as detected with solo ABC-treated sections. Moreover, the ABC-DAB/H2O2 mediated pattern corresponded to the spatial orientation of Müller cells as identified by GFAP immunostaining. These findings indicate the presence of endogenous biotin in Müller cells and calls for caution in the application of the ABC method in immunotechniques in retinal research.

Animals↗

Quantitative analysis of cell columns in the cerebral cortex.

We present a quantified imaging method that describes the cell column in mammalian cortex. The minicolumn is an ideal template with which to examine cortical organization because it is a basic unit of function, complete in itself, which interacts with adjacent and distance columns to form more complex levels of organization. The subtle details of columnar anatomy should reflect physiological changes that have occurred in evolution as well as those that might be caused by pathologies in the brain. In this semiautomatic method, images of Nissl-stained tissue are digitized or scanned into a computer imaging system. The software detects the presence of cell columns and describes details of their morphology and of the surrounding space. Columns are detected automatically on the basis of cell-poor and cell-rich areas using a Gaussian distribution. A line is fit to the cell centers by least squares analysis. The line becomes the center of the column from which the precise location of every cell can be measured. On this basis several algorithms describe the distribution of cells from the center line and in relation to the available surrounding space. Other algorithms use cluster analyses to determine the spatial orientation of every column.

Algorithms↗

Oxiracetam prevents the MK-801 induced amnesia for the elevated plus-maze in mice.

We investigated the effect of the nootropic substance oxiracetam on the impairment of memory induced in mice by the non-competitive NMDA antagonist MK-801. Memory capacities of animals having different experience were evaluated using the elevated plus-maze test. Oxiracetam was injected immediately after the acquisition session(s), MK-801 was given 30 min before the retention session which followed 24 h after the acquisition session(s). In slightly experienced animals (Section 3.1), oxiracetam (3 and 30 mg/kg, s.c.) prevented MK-801 (0.15 mg/kg, i.p.) induced memory deficits characterized by a prolongation of the transfer latency. In well-trained animals (Section 3.2), oxiracetam (30 mg/kg, s.c.) attenuated MK-801 (0.15,0. 25 and 0.4 mg/kg, i.p.) induced amnesia for a spatial orientation in the elevated plus-maze. These results show that oxiracetam interacted with the glutamatergic NMDA receptor system and forestalled the impairment of retrieval of long-term memory. The results also justify the usage of the elevated plus-maze method in the evaluation of potential anti-amnesic or nootropic drugs.

Amnesia↗

MK-801 induced amnesia for the elevated plus-maze in mice.

MK-801, a non-competitive NMDA receptor antagonist, has been shown to have amnesic properties in animal models. The purpose of the present study was to examine potential amnesic effects of MK-801 in mice using the modified elevated plus-maze paradigm. An animal was placed on the distal end of an open arm, and the transfer latency, i.e. the time in which it moves to the enclosed arm, was measured. Four different experimental schedules (i.e. the combination of the treatment and the testing) were used: MK-801 (0.075, 0.15, 0.25 and 0.4 mg/kg or saline) were given (a) 30 min before the acquisition session, (b) immediately after the acquisition session, (c) 60 min after the acquisition session, and (d) 30 min before the retention session. The retention session always followed 24 h after the acquisition session. Analysis of data showed a significant shortening of the transfer latency in saline-treated animals during the retention session. Further, MK-801 (at the dose range of 0.15--0.4 mg/kg) administered before and immediately after the acquisition session as well as before the retention session prolonged the transfer latency during the retention session. In fact, transfer latencies in MK-801 treated mice did not differ from those measured during the acquisition session. Thus, prolongation of the transfer latency in MK-801-treated mice indicates deficits in 'memorization' processes. On the contrary, MK-801 given 60 min after the acquisition session failed to increase the transfer latency, which suggests that the memory trace was sufficiently consolidated at this time. Based on the present results, the glutamatergic NMDA receptor mechanisms play an important role in a spatial orientation of mice placed on the elevated plus-maze.

Amnesia↗

Tiagabine, a gamma-amino-butyric acid transporter inhibitor impairs spatial learning of rats in the Morris water-maze.

gamma-Amino-butyric acid (GABA) is cleaved from the synaptic cleft by uptake via specific transporters. Inhibition of such transporters increases the effectiveness of physiologically released GABA. Increased GABAergic neurotransmission has an impact on learning and memory. Therefore, effects of tiagabine, a GABA-transporter inhibitor, were investigated on spatial orientation in the Morris water-maze. Rats were given four training trials per day for 4 days and a probe trial without platform on the 5th day. Compared to saline treated rats, rats treated daily with 20 mg/kg tiagabine showed impaired learning during the acquisition trials. Retrieval was impaired in rats treated only at the probe trial with tiagabine. These results further elucidate the role of GABA in learning and memory.

Animals↗

The effects of combined superior temporal polysensory area and frontal eye field lesions on eye movements in the macaque monkey.

We previously found [42] that lesions of the superior temporal polysensory area (STP) cause temporary deficits in the production of eye movements. In order to both define regions participating in the ensuing recovery and to further explore the cortical control of eye movements, we examined the effects of addition of frontal eye field (FEF) lesions to STP lesions, on visual fixation, saccadic eye movements, and smooth pursuit eye movements. Three monkeys received bilateral STP lesions followed by a FEF lesion and as a control, an additional monkey received a bilateral inferior temporal cortex (IT) lesion followed by a FEF lesion. All animals had a profound impairment in foveating the central fixation point. This impairment was completely eliminated by turning on a dim light in the testing chamber. Large neglect-like impairments in making saccades were only seen after combined STP and FEF lesions. Impairments in making smooth pursuit eye movements after combined lesions of STP and FEF were larger than those seen after STP lesions but within the range of deficits that have been reported after FEF lesions alone. The impairment of visual fixation in darkness and the lack of impairment under conditions of dim illumination appear to reflect a specific role for the FEF in spatial orientation in the absence of visual landmarks. The FEF also appears to play a more critical role than STP in smooth pursuit. By contrast, STP and the FEF appear to work cooperatively with respect to the production of saccades. We suggest that cortical oculomotor control can flow either through the midbrain or through the FEF and that the FEF pathway is specifically involved in tasks with a discontiguity between the stimuli and the behavioral response while the midbrain pathways are preferentially involved in more stimulus-driven eye movements.

Animals↗

Concurrent administration of subeffective doses of scopolamine and MK-801 produces a short-term amnesia for the elevated plus-maze in mice.

Amnesic properties of scopolamine, a muscarinic receptor antagonist, and MK-801, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, were evaluated in mice by means of the elevated plus-maze test. In this test, transfer latency, the time mice took to move from the open arm to the enclosed arm, was used as an index of learning and memory. The 3-day pretreatment training dramatically decreased transfer latencies. On the 4th day, the retention trial was performed 30 min after the intraperitoneal injection of scopolamine (Experiment 1) or MK-801 (Experiment 2). The doses of 0.25 and 0.5 mg/kg of scopolamine as well as the doses of 0.1, 0.15, 0.2 and 0.4 mg/kg of MK-801 significantly prolonged the transfer latency as compared with both that in the saline-treated group and that measured on the 3rd day. In Experiment 3, subthreshold doses of these two drugs given in combination (which were ineffective when given alone: scopolamine 0.25 mg/kg, MK-801 0.075 mg/kg) significantly prolonged the transfer latency on the fourth day. However, an amnesic effect of scopolamine plus MK-801 was transient. On the 5th day, no differences in the transfer latency were found. This finding clearly indicates that there is a close relationship between cholinergic and glutamatergic systems and that both systems play an important role in a spatial orientation of mice on the elevated plus-maze.

Amnesia↗

Shift in the performance of 24-month-old Wistar rats in the Morris water escape task: a comparison across 36 experiments.

Spatial discrimination learning in aged rats serves as an animal model of cognitive aging. We assessed the replicability of spatial discrimination performance in the standard Morris water escape task. To this end the learning curves and the performance in a probe trial of 24-month-old outbred Wistar (HsdWin:Wu) control rats from 36 experiments were compared. These experiments had been performed at our laboratory under strictly controlled conditions over a period of 71 weeks. There was a very high variability in the learning curves between experiments. The initial performance level, i.e. the performance during the first session, did not change systematically across the 36 experiments. In contrast, the final performance level, i.e. the level reached in the fifth training session, decreased over the 71 week period, when the platform escape latency and the distance swam to reach the platform, measured as number of line crossings, were considered. In the last experiments of the series, learning curves were no longer seen: the rats did not improve their performance across the acquisition sessions. By contrast, the swimming speed and, in the probe trial, the bias for the quadrant where the platform had been positioned during training, did not change. This indicates that a decrease across experiments occurred predominantly with respect to spatial orientation performance, whereas the motor performance appeared to be unchanged. Explanations for this observation, such as differences in viability between shipments and the possible occurrence of genetic drift, are discussed.

Aging↗

Cellular effects of leishmanial tubulin inhibitors on L. donovani.

To aid our investigation of tubulin as an antileishmanial drug target, the effects of the mammalian antimicrotubule agents ansamitocin P3, taxol, and hemiasterlin on Leishmania donovani promastigotes were described. These drugs affected the assembly of purified leishmanial tubulin and inhibited the growth of L. donovani promastigotes at micromolar concentrations. When promastigotes were treated with these agents, mitotic partitioning of nuclear DNA and cytokinesis were usually inhibited. The spatial orientation of kinetoplasts was often disturbed, suggesting a role for microtubules in the segregation of these organelles during mitosis. Aberrant cell types produced in drug-treated cultures included parasites with one nucleus and two geometrically distinct kinetoplasts, parasites with multiple kinetoplasts, and cytoplasts containing a kinetoplast but no nucleus. A subset of unique cell types, parasites containing two nuclei, a spindle fiber, and two geometrically distinct kinetoplasts, were observed in hemiasterlin-treated cultures. Flow cytometric analysis of L. donovani promastigotes treated with these three drugs indicated a dramatic shift toward the G2 + M phase of the cell cycle, with some cells containing four times the amount of DNA present in G1. These results were used to evaluate the cellular effects of WR85915, an aromatic thiocyanate with in vitro antileishmanial and anti-tubulin activity, on L. donovani. Treatment of parasites with WR85915 did not produce the unusual cell types described above and did not cause the accumulation of parasites in G2 + M, suggesting that WR85915 acts on target(s) in Leishmania in addition to tubulin. These studies validate tubulin as a suitable antileishmanial drug target and provide criteria to assess the cellular mechanism of action of new candidate antileishmanial agents.

Animals↗