The Role of Tissue Geometry in Spinal Cord Regeneration.

Unlike peripheral nerves, axonal regeneration is limited following injury to the spinal cord. While there may be reduced regenerative potential of injured neurons, the central nervous system (CNS) white matter environment appears to be more significant in limiting regrowth. Several factors may inhibit regeneration, and their neutralization can modestly enhance regrowth. However, most investigations have not considered the cytoarchitecture of spinal cord white matter. Several lines of investigation demonstrate that axonal regeneration is enhanced by maintaining, repairing, or reconstituting the parallel geometry of the spinal cord white matter. In this review, we focus on environmental factors that have been implicated as putative inhibitors of axonal regeneration and the evidence that their organization may be an important determinant in whether they inhibit or promote regeneration. Consideration of tissue geometry may be important for developing successful strategies to promote spinal cord regeneration.

Segregated neural explants exhibit co-oriented, asymmetric, neurite outgrowth.

Explants of embryonic chick sympathetic and sensory ganglia were found to exhibit asymmetric radial outgrowth of neurites under standard culture conditions with or without exogenous Nerve Growth Factor [NGF]. Opposing sides of an explant exhibited: a) differences in neurite length and, b) differences in neurite morphology. Strikingly, this asymmetry exhibited co-orientation among segregated, neighboring explants. The underlying mechanism(s) of the asymmetry and its co-orientation are not known but appear to depend on cell clustering because dissociated sympathetic neurons do not exhibit co-orientation whereas re-aggregated clusters of cells do. This emergent behavior may be similar to the community effect described in other cell types. If a similar phenomenon exists in the embryo, or in maturity, it may contribute to the establishment of proper orientation of neurite outgrowth during development and/or injury-induced neuronal plasticity.

Pia Mater Significantly Contributes to Spinal Cord Intraparenchymal Pressure in a Simulated Model of Edema.

STUDY DESIGN: Intraparenchymal pressure (IPP) measurements in an in vitro cadaveric model of CNS edema. OBJECTIVE: To assess the contribution of pia mater to IPP and the effect of piotomy. SUMMARY OF BACKGROUND DATA: Multicenter randomized control trials have shown that decompression with durotomy/duroplasty significantly decreases intracranial pressure (ICP). There is a paucity of evidence regarding the effectiveness of decompression of the spinal cord by piotomy. METHODS: The supratentorial brain and spinal cord were removed from six fresh cadavers. Dura and arachnoid mater were removed. ICP monitors were placed bilaterally in the frontal and parietal lobes, and centrally in the cervical and thoracic spinal cord. To simulate edema, specimens were submerged in hypotonic solution. IPP was recorded for 5 days. A complete dorsal midline piotomy was performed on the spinal cord and resulting IPP was recorded. RESULTS: Brain and spinal cord both increased in weight. IPP significantly increased in both brain and spinal cord. The IPP increase within the spinal cord was substantially greater (averages: all four lobes = 4.0 mm Hg; cervical = 73.7 mm Hg; thoracic = 49.3 mm Hg). After piotomy, cervical and thoracic spinal cord IPP decreased immediately (avg. postpiotomy IPP = 9.7 and 10.3, respectively). CONCLUSION: There were differential effects on brain and spinal cord IPP. Brain IPP increased only slightly, possibly because of the absence of the cranium and dura mater. In contrast, spinal cord IPP increased substantially even in the absence of the laminae, dura, and arachnoid mater. Piotomy immediately and dramatically reduced spinal cord IPP. These data are consistent with the hypothesis that spinal cord IPP is primarily dependent on constraints imposed by the pia mater. Conversely, in the absence of the cranium and dura mater, the sulci may permit the pia-invested brain to better accommodate edema without significant increases in IPP. LEVEL OF EVIDENCE: N/A.

Spinal cord intramedullary pressure in thoracic scoliotic deformity: a cadaveric study.

STUDY DESIGN: In vitro cadaveric study of thoracic spinal cord intramedullary pressure (IMP) in scoliotic deformity. OBJECTIVE: To define the relationship between thoracic scoliotic deformity and spinal cord IMP. SUMMARY OF BACKGROUND DATA: Clinical studies of patients with thoracic scoliosis without other spinal pathology (spinal stenosis, etc.) have rarely reported an associated thoracic myelopathy. Previous clinical and cadaveric studies of kyphosis have reported associated myelopathy and increased spinal cord IMP. We sought to determine if IMP changes in response to main thoracic scoliotic deformity. METHODS: In 6 fresh-frozen cadavers, a progressive main thoracic scoliotic deformity was created. Cadavers were positioned sitting with physiological spinal alignment, head stabilized using a skull clamp and spine segmentally instrumented from occiput to L3. The T3-T4 ligamentum flavum was removed, dura opened, and 3 pressure sensors were advanced caudally to T4-T5, T7-T8, and T10-T11 within the cord parenchyma. A step-wise main thoracic scoliotic deformity was then induced by sequentially releasing and retightening the skull clamp while coronally bending, concavity compressing, and convexity distracting posterior segmental instrumentation, allowing closure of lateral segmental osteotomies. After each step, fluoroscopic images and pressure measurements were obtained; the T4-T11 coronal Cobb angle was measured. RESULTS: Induction of main thoracic scoliosis did not significantly increase IMP. The mean main thoracic maximal scoliotic deformity created was 77° ± 2° (range: 71°-84°). At maximal deformity, the mean ΔIMP at T4-T5, T7-T8, T10-T11 was 2.2 ± 1.9 mm Hg, 1.0 ± 0.7 mm Hg, and 1.0 ± 0.8 mm Hg, respectively. CONCLUSION: In this cadaveric study, main thoracic scoliotic deformity did not significantly increase thoracic IMP. This correlates with clinical presentation such that clinical studies of patients with thoracic scoliosis without other spinal pathology have rarely reported an associated thoracic myelopathy with the thoracic scoliosis. This study helps explain the relative absence of myelopathy in isolated main thoracic coronal plane deformity. LEVEL OF EVIDENCE: 5.

Performance evaluation of a whole blood propofol analyser.

The authors evaluated an analyser for the determination of propofol concentrations in whole blood. The Pelorus 1000 (Sphere Medical) measures propofol concentrations in around 5 min without the requirement for sample preparation. The performance of the analyser was characterised with respect to linearity, precision in control solutions and whole blood and method comparison to an HPLC based reference method. In addition, the effects of substances considered to potentially affect the assay method were investigated. The analyser was found to be linear up to 12 µg/ml (R2 = 0.9993), with a lower limit of quantification of 0.75 µg/ml. Total within device imprecision in control solutions was 0.11 µg/ml at 5.32 µg/ml and 0.17 µg/ml at 10.3 µg/ml. Within run precision in whole blood was 0.04 µg/ml at 2.84 µg/ml and 0.08 µg/ml at 6.68 µg/ml and for the reference method was 0.06 µg/ml and 0.12 µg/ml respectively. In comparison to the reference method, the overall bias of the Pelorus 1000 system over the range is estimated to be 0.15 µg/ml (95% confidence interval -0.11-0.41 µg/ml). The only cross interference of note is to a highly elevated level of conjugated bilirubin, while low haematocrit levels lead to a 0.13 µg/ml under reading with respect to the HPLC reference. The system fulfils the requirements for measurement of propofol concentrations in whole blood samples with precision and accuracy suitable for elucidating propofol pharmacokinetics at clinically relevant concentrations. With no requirement for sample preparation and a fast time to results, the analyser opens up the possibility of studies to measure and respond to blood propofol concentrations in patients in close to real time.

Laminectomy, durotomy, and piotomy effects on spinal cord intramedullary pressure in severe cervical and thoracic kyphotic deformity: a cadaveric study.

OBJECT: Previous studies have shown that cervical and thoracic kyphotic deformity increases spinal cord intramedullary pressure (IMP). Using a cadaveric model, the authors investigated whether posterior decompression can adequately decrease elevated IMP in severe cervical and thoracic kyphotic deformities. METHODS: Using an established cadaveric model, a kyphotic deformity was created in 16 fresh human cadavers (8 cervical and 8 thoracic). A single-level rostral laminotomy and durotomy were performed to place intraparenchymal pressure monitors in the spinal cord at C-2, C4-5, and C-7 in the cervical study group and at T4-5, T7-8, and T11-12 in the thoracic study group. Intramedullary pressure was recorded at maximal kyphosis. Posterior laminar, dural, and pial decompressions were performed while IMP was monitored. In 2 additional cadavers (1 cervical and 1 thoracic), a kyphotic deformity was created and then corrected. RESULTS: The creation of the cervical and thoracic kyphotic deformities resulted in significant increases in IMP. The mean increase in cervical and thoracic IMP (change in IMP [ΔIMP]) for all monitored levels was 37.8 ± 7.9 and 46.4 ± 6.4 mm Hg, respectively. After laminectomies were performed, the mean cervical and thoracic IMP was reduced by 22.5% and 18.5%, respectively. After midsagittal durotomies were performed, the mean cervical and thoracic IMP was reduced by 62.8% and 69.9%, respectively. After midsagittal piotomies were performed, the mean cervical and thoracic IMP was reduced by 91.3% and 105.9%, respectively. In 2 cadavers in which a kyphotic deformity was created and then corrected, the ΔIMP increased with the creation of the deformity and returned to zero at all levels when the deformity was corrected. CONCLUSIONS: In this cadaveric study, laminar decompression reduced ΔIMP by approximately 15%-25%, while correction of the kyphotic deformity returned ΔIMP to zero. This study helps explain the pathophysiology of myelopathy in kyphotic deformity and the failure of laminectomy alone for cervical and thoracic kyphotic deformities with myelopathy. In addition, the study emphasizes the need for correction of deformity during operative treatment of kyphotic deformity, the need for maintaining adequate intraoperative blood pressure during operative treatment, and the higher risk of spinal cord injury associated with operative treatment of kyphotic deformity.

Spinal cord intramedullary pressure in thoracic kyphotic deformity: a cadaveric study.

STUDY DESIGN: In vitro cadaveric study of thoracic spinal cord intramedullary pressure (IMP) in kyphotic deformity. OBJECTIVE: To define the relationship between thoracic spinal kyphotic deformity and spinal cord IMP. SUMMARY OF BACKGROUND DATA: Previous studies of asymptomatic volunteers have revealed that there is wide variation in regional sagittal neutral upright thoracic spinal alignment with "normal" thoracic T4-T12 kyphosis ranging up to approximately +69° for 98.5% of the asymptomatic adult population. We sought to determine whether IMP changes in response to increasing thoracic kyphosis. METHODS: In 8 fresh-frozen cadavers, a progressive kyphotic deformity was created. Cadavers were positioned sitting with physiological thoracic kyphosis, head stabilized using a skull clamp, and spine segmentally instrumented from occiput to L2. The T3-T4 ligamentum flavum was removed, dura opened, and 3 pressure sensors were advanced caudally to T4-T5, T7-T8, and T11-T12 within the cord parenchyma. A stepwise thoracic kyphotic deformity was then induced by sequentially releasing and retightening the skull clamp while distracting posterior short segment rods and closing anterior segmental osteotomies. After each step, fluoroscopic images and pressure measurements were obtained; the T4-T12 Cobb angle was measured. RESULTS: Minor IMP increases of 2 to 5 mm Hg were observed at 1 or more spinal cord levels in 1 of 8 cadavers when the Cobb angle was less than +51° and in 4 of 8 cadavers when the angle was more than +51° and less than +63°. For Cobb angles more than +51° and less than +63°, a statistically significant, minor increase in IMP was detected at the T7-T8 level only (P = 0.02). At Cobb angles exceeding +63°, ΔIMP progressively increased at 1 or more spinal cord levels in 8 of 8 cadavers. Cobb angles ranging from +63° to +149° resulted in statistically significant increases in IMP ranging to more than 50 mm Hg. ΔIMP did not correlate with segmental spinal canal diameter (stenosis). CONCLUSION: Thoracic kyphosis less than +51° resulted in no meaningful increase in IMP, whereas kyphosis measuring +51° to +63° resulted in minor increases in IMP. After the thoracic kyphosis exceeded +63°, IMP increased significantly. ΔIMP with spinal alignment may help explain the wide range of "normal" thoracic neutral upright sagittal alignment in studies of asymptomatic adult individuals and may help further define thoracic kyphotic deformity.

Spinal cord intramedullary pressure in cervical kyphotic deformity: a cadaveric study.

STUDY DESIGN: In vitro cadaveric study of cervical spinal cord intramedullary pressure (IMP) in kyphotic deformity. OBJECTIVE: To define the relationship between cervical spinal kyphotic deformity and spinal cord IMP. SUMMARY OF BACKGROUND DATA: Previous studies of asymptomatic volunteers have revealed that the greatest variation in regional sagittal neutral upright spinal alignment occurs in the cervical spine with "normal" alignment ranging up to +15 to +20° kyphosis. We sought to determine whether IMP changes in response to increasing cervical kyphosis. METHODS: In eight fresh-frozen cadavers, a progressive kyphotic deformity was created. Cadavers were positioned sitting with cervical lordosis, with head stabilized using a skull clamp. The C1 posterior arch was removed, dura was opened, and three pressure sensors were advanced caudally to C7, C4-C5, and C2 within the cord parenchyma. A stepwise kyphotic deformity was then induced by sequentially releasing and retightening the skull clamp while distracting posterior short segment rods and closing anterior segmental osteotomies. After each step, fluoroscopic images and pressure measurements were obtained. The C2-C7 Gore angle and horizontal displacement of the odontoid plumb line relative to C7 (C2-C7 sagittal vertical axis [SVA]) were measured. RESULTS: Minor IMP increases of 2 to 5 mm Hg were observed at one or more spinal cord levels in one of eight cadavers when the Gore angle was <+7.5° and in three of eight cadavers when the Gore angle was >+7.5° and <+21°. At Gore angles exceeding +21°, change in pressure (ΔIMP) progressively increased at one or more spinal cord levels in eight of eight cadavers. Gore angles ranging from +21° to +78° resulted in statistically significant increases in IMP ranging to >50 mm Hg, as did C2-C7 SVA >+75 mm. ΔIMP did not correlate with segmental spinal canal diameter (stenosis). CONCLUSION: Cervical lordosis and kyphosis less than +7.5° resulted in no meaningful increase in IMP. Minor cervical kyphosis measuring +7.5° to +21° resulted in 2 to 5 mm Hg increases in IMP. As the cervical kyphotic deformity exceeded +21°, IMP increased significantly. ΔIMP with spinal alignment may help to explain the wide range of "normal" cervical neutral upright sagittal alignment in studies of asymptomatic individuals and may help further define cervical kyphotic deformity.

Virtual preoperative measurement and surgical manipulation of sagittal spinal alignment using a novel research and educational software program.

Understanding regional as well as global spinal alignment is increasingly recognized as important for the spine surgeon. A novel software program for virtual preoperative measurement and surgical manipulation of sagittal spinal alignment was developed to provide a research and educational tool for spine surgeons. This first-generation software program provides tools to measure sagittal spinal alignment from the occiput to the pelvis, and to allow for virtual surgical manipulation of sagittal spinal alignment. The software was developed in conjunction with Clifton Labs, Inc. Photographs and radiographs were imported into the software program, and a 2D virtual spine was constructed from the images. The software then measured regional and global sagittal spinal alignment from the virtual spine construct, showing the user how to perform the measurements. After measuring alignment, the program allowed for virtual surgical manipulation, simulating surgical procedures such as interbody fusion, facet osteotomy, pedicle subtraction osteotomy, and reduction of spondylolisthesis, as well as allowing for rotation of the pelvis on the hip axis. Following virtual manipulation, the program remeasured regional and global sagittal spinal alignment. Computer software can be used to measure and manipulate sagittal spinal alignment virtually, providing a new research and educational tool. In the future, more comprehensive programs may allow for measurement and interaction in the coronal, axial, and sagittal planes.

Major neurologic deficit immediately after adult spinal surgery: incidence and etiology over 10 years at a single training institution.

STUDY DESIGN: Retrospective study of adult patients who underwent spinal surgery over a 10-year period at a single institution. OBJECTIVE: New onset postoperative paralysis remains one of the most feared complications of spinal surgery. The goal of this study was to determine the incidence and etiology of new onset major neurologic deficit immediately after adult spinal surgery. SUMMARY OF BACKGROUND DATA: Previous studies, focusing on specific disease entities, have shown incidence rates of significant spinal cord or cauda equina injury after spinal surgery ranging from approximately 0% to 2%. METHODS: The authors reviewed the quality assurance records for adult patients who underwent spinal surgery over a 10-year period (July 1, 1996 to June 30, 2006) by surgeons in the Department of Neurosurgery, University of Cincinnati College of Medicine at hospitals affiliated with the neurologic surgery residency program. Patients with new onset major neurologic deficit immediately after spinal surgery were identified. RESULTS: Of 11,817 adult spinal operations, 21 patients experienced new onset major neurologic deficit immediately after spinal surgery, yielding an overall incidence of 0.178%; in the cervical spine 0.293%, thoracic spine 0.488%, and lumbar/sacral spine 0.0745%. The difference in incidence between spinal regions was statistically significant (P = 0.00343). The etiology of the neurologic deficits was confirmed with reoperation and/or postoperative imaging studies: epidural hematoma in 8 patients, inadequate decompression in 5 patients, presumed vascular compromise in 4 patients, graft/cage dislodgement in 2 patients, and presumed surgical trauma in 2 patients. Placement of spinal instrumentation was performed in 12 of 21 patients (57.1%) and was associated with a significantly higher risk of new onset major neurologic deficit immediately after spinal surgery (P = 0.022). CONCLUSIONS: The incidence of new onset major neurologic deficit immediately after adult spinal surgery is low. Epidural hematoma and inadequate decompression were the most common etiologies in this series of patients.

Leukocyte analysis and differentiation using high speed microfluidic single cell impedance cytometry.

Miniature high speed label-free cell analysis systems have yet to be developed, but have the potential to deliver fast, inexpensive and simple full blood cell analysis systems that could be used routinely in clinical practice. We demonstrate a microfluidic single cell impedance cytometer that performs a white blood cell differential count. The device consists of a microfluidic chip with micro-electrodes that measure the impedance of single cells at two frequencies. Human blood, treated with saponin/formic acid to lyse erythrocytes, flows through the device and a complete blood count is performed in a few minutes. Verification of cell dielectric parameters was performed by simultaneously measuring fluorescence from CD antibody-conjugated cells. This enabled direct correlation of impedance signals from individual cells with phenotype. Tests with patient samples showed 95% correlation against commercial (optical/Coulter) blood analysis equipment, demonstrating the potential clinical utility of the impedance microcytometer for a point-of-care blood analysis system.

A structural study of the interaction between the Dr haemagglutinin DraE and derivatives of chloramphenicol.

Dr adhesins are expressed on the surface of uropathogenic and diffusely adherent strains of Escherichia coli. The major adhesin subunit (DraE/AfaE) of these organelles mediates attachment of the bacterium to the surface of the host cell and possibly intracellular invasion through its recognition of the complement regulator decay-accelerating factor (DAF) and/or members of the carcinoembryonic antigen (CEA) family. The adhesin subunit of the Dr haemagglutinin, a Dr-family member, additionally binds type IV collagen and is inhibited in all its receptor interactions by the antibiotic chloramphenicol (CLM). In this study, previous structural work is built upon by reporting the X-ray structures of DraE bound to two chloramphenicol derivatives: chloramphenicol succinate (CLS) and bromamphenicol (BRM). The CLS structure demonstrates that acylation of the 3-hydroxyl group of CLM with succinyl does not significantly perturb the mode of binding, while the BRM structure implies that the binding pocket is able to accommodate bulkier substituents on the N-acyl group. It is concluded that modifications of the 3-hydroxyl group would generate a potent Dr haemagglutinin inhibitor that would not cause the toxic side effects that are associated with the normal bacteriostatic activity of CLM.

Spinal deformity: a new classification derived from neutral upright spinal alignment measurements in asymptomatic juvenile, adolescent, adult, and geriatric individuals.

OBJECTIVE: In this literature review, the authors analyze data from previously published studies that evaluated neutral upright spinal alignment (NUSA) from the occiput to the pelvis in asymptomatic individuals. Based on the data for NUSA in asymptomatic volunteers, a new classification is proposed for spinal deformity. METHODS: A review of the English literature was conducted to identify studies evaluating NUSA from the occiput to the pelvis in asymptomatic juvenile, adolescent, adult, and geriatric volunteers. From the literature review, 17 angles and displacements were selected to depict neutral upright coronal and axial spinal alignment, and 21 angles and displacements were selected to depict neutral upright sagittal spinal alignment. Pooled estimates of the mean and variance were calculated for the angles and displacements from the articles that met inclusion criteria. A new classification of spinal deformity was then developed based on age-dependent NUSA; spinal abnormality; deformity curve location, pattern, magnitude, and flexibility; and global spinal alignment. RESULTS: Despite a wide variation in the regional curves from the occiput to the pelvis in asymptomatic volunteers, global spinal alignment is maintained in a narrow range for preservation of horizontal gaze and balance of the spine over the pelvis and femoral heads. CONCLUSION: A new classification of spinal deformity is proposed that provides a structure for defining deformity of all patient ages and spinal abnormalities.

A simple, efficient tool for assessment of mice after unilateral cortex injury.

A refined battery of neurological tests, SNAP (Simple Neuroassessment of Asymmetric Impairment), was developed and validated to efficiently assess neurological deficits induced in a mouse model of traumatic brain injury. Four to 7-month old mice were subjected to unilateral controlled cortical impact or sham injury (craniectomy only). Several behavioral tests (SNAP, beam walk, foot fault, and water maze) were used to assess functional deficits. SNAP was unique among these in that it required no expensive equipment and was performed in less than 5 min per mouse. SNAP demonstrated a high level of sensitivity and specificity as determined by receiver-operator characteristics curve analysis. Interrater reliability was good, as determined by Cohen's Kappa method and by comparing the sensitivity and specificity across various raters. SNAP detected deficits in proprioception, visual fields, and motor strength in brain-injured mice at 3 days, and was sensitive enough to detect magnitude and recovery of injury. The contribution of individual battery components changed as a function of time after injury, however, each was important to the overall SNAP score. SNAP provided a sensitive, reliable, time-efficient and cost-effective means of assessing neurological deficits in mice after unilateral brain injury.

Global expression of NGF promotes sympathetic axonal growth in CNS white matter but does not alter its parallel orientation.

Axonal regeneration is normally limited after injuries to CNS white matter. Infusion of neurotrophins has been successful in promoting regenerative growth through injured white matter but this growth generally fails to extend beyond the infusion site. These observations are consistent with a chemotropic effect of these factors on axonal growth and support the prevailing view that neurotrophin-induced axonal regeneration requires the use of gradients, i.e., gradually increasing neurotrophin levels along the target fiber tract. To examine the potential of global overexpression of neurotrophins to promote, and/or modify the orientation of, regenerative axonal growth within white matter, we grafted nerve growth factor (NGF) responsive neurons into the corpus callosum of transgenic mice overexpressing NGF throughout the CNS under control of the promoter for glial fibrillary acidic protein. One week later, glial fibrillary acidic protein and chondroitin sulfate proteoglycan immunoreactivity increased within injured white matter around the grafts. NGF levels were significantly higher in the brains of transgenic compared with non-transgenic mice and further elevated within injury sites compared with the homotypic region of the non-injured side. Although there was minimal outgrowth from neurons grafted into non-transgenic mice, extensive parallel axonal regeneration had occurred within the corpus callosum up to 1.5 mm beyond the astrogliotic scar (the site of maximum NGF expression) in transgenic mice. These results demonstrate that global overexpression of neurotrophins does not override the constraints limiting regenerative growth to parallel orientations and suggest that such factors need not be presented as positive gradients to promote axonal regeneration within white matter.

Decay-accelerating factor must bind both components of the complement alternative pathway C3 convertase to mediate efficient decay.

Decay-accelerating factor (DAF; CD55) inhibits the complement (C) cascade by dissociating the multimolecular C3 convertase enzymes central to amplification. We have previously demonstrated using surface plasmon resonance (Biacore International) that DAF mediates decay of the alternative pathway C3 convertase, C3bBb, but not of the inactive proenzyme, C3bB, and have shown that the major site of interaction is with the larger cleavage subunit factor B (Bb) subunit. In this study, we dissect these interactions and demonstrate that the second short consensus repeat (SCR) domain of DAF (SCR2) interacts only with Bb, whereas SCR4 interacts with C3b. Despite earlier studies that found SCR3 to be critical to DAF activity, we find that SCR3 does not directly interact with either subunit. Furthermore, we demonstrate that properdin, a positive regulator of the alternative pathway, does not directly interact with DAF. Extending from studies of binding to decay-accelerating activity, we show that truncated forms of DAF consisting of SCRs 2 and 3 bind the convertase stably via SCR2-Bb interactions but have little functional activity. In contrast, an SCR34 construct mediates decay acceleration, presumably due to SCR4-C3b interactions demonstrated above, because SCR3 alone has no binding or functional effect. We propose that DAF interacts with C3bBb through major sites in SCR2 and SCR4. Binding to Bb via SCR2 increases avidity of binding, concentrating DAF on the active convertase, whereas more transient interactions through SCR4 with C3b directly mediate decay acceleration. These data provide new insights into the mechanisms involved in C3 convertase decay by DAF.

Structural and functional insights into the interaction of echoviruses and decay-accelerating factor.

Many enteroviruses bind to the complement control protein decay-accelerating factor (DAF) to facilitate cell entry. We present here a structure for echovirus (EV) type 12 bound to DAF using cryo-negative stain transmission electron microscopy and three-dimensional image reconstruction to 16-A resolution, which we interpreted using the atomic structures of EV11 and DAF. DAF binds to a hypervariable region of the capsid close to the 2-fold symmetry axes in an interaction that involves mostly the short consensus repeat 3 domain of DAF and the capsid protein VP2. A bulge in the density for the short consensus repeat 3 domain suggests that a loop at residues 174-180 rearranges to prevent steric collision between closely packed molecules at the 2-fold symmetry axes. Detailed analysis of receptor interactions between a variety of echoviruses and DAF using surface plasmon resonance and comparison of this structure (and our previous work; Bhella, D., Goodfellow, I. G., Roversi, P., Pettigrew, D., Chaudhry, Y., Evans, D. J., and Lea, S. M. (2004) J. Biol. Chem. 279, 8325-8332) with reconstructions published for EV7 bound to DAF support major differences in receptor recognition among these viruses. However, comparison of the electron density for the two virus.receptor complexes (rather than comparisons of the pseudo-atomic models derived from fitting the coordinates into these densities) suggests that the dramatic differences in interaction affinities/specificities may arise from relatively subtle structural differences rather than from large-scale repositioning of the receptor with respect to the virus surface.

Hydrophilic domain II of Escherichia coli Dr fimbriae facilitates cell invasion.

Uropathogenic and diarrheal Escherichia coli strains expressing adhesins of the Dr family bind to decay-accelerating factor, invade epithelial cells, preferentially infect children and pregnant women, and may be associated with chronic or recurrent infections. Thus far, no fimbrial domain(s) that facilitates cell invasion has been identified. We used alanine scanning mutagenesis to replace selected amino acids in hydrophilic domain II of the structural fimbrial subunit DraE and evaluated recombinant mutant DraE for attachment, invasion, and intracellular compartmentalization. The mutation of amino acids V28, T31, G33, Q34, T36, and P40 of DraE reduced or abolished HeLa cell invasion but did not affect attachment. Electron micrographs showed a stepwise entry and fusion of vacuoles containing Escherichia coli mutants T36A and Q34A or corresponding beads with lysosomes, whereas vacuoles with wild-type Dr adhesin showed no fusion. Mutants T31A and Q34A, which were deficient in invasion, appeared to display a reduced capacity for clustering decay-accelerating factor. Our findings suggest that hydrophilic domain II may be involved in cell entry. These data are consistent with the interpretation that in HeLa cells the binding and invasion phenotypes of Dr fimbriae may be separated.

Dynamic properties of regulatory motifs associated with induction of three temporal domains of memory in aplysia.

A model was developed to examine dynamical properties of regulatory motifs correlated with different temporal domains of memory. The model represents short-, intermediate-, and long-term phases of protein kinase A (PKA) activation, which appear related to corresponding phases of facilitation of the Aplysia sensorimotor synapse. The model also represents phosphorylation of the transcription factor CREB1 by PKA and consequent induction of the immediate-early gene Aplysia ubiquitin hydrolase (Ap-uch), which is essential for long-term synaptic facilitation (LTF). Simulations suggest mechanisms responsible for differing profiles of synaptic facilitation following massed vs. spaced exposures to 5-HT, and suggest a novel regulatory motif (gated positive feedback) is important for LTF. Simulations suggest zero-order ultrasensitivity may underlie a requirement of a threshold number of exposures to 5-HT for LTF induction. The model makes predictions for the dynamics of PKA activation and Ap-uch induction when MAP kinase is activated, or when repression of Ap-uch is relieved by inhibiting the transcription factor CREB2. This model may therefore be useful for understanding processes underlying memory formation in Aplysia and other systems.

High resolution studies of the Afa/Dr adhesin DraE and its interaction with chloramphenicol.

Pathogenic Escherichia coli expressing Afa/Dr adhesins are able to cause both urinary tract and diarrheal infections. The Afa/Dr adhesins confer adherence to epithelial cells via interactions with the human complement regulating protein, decay accelerating factor (DAF or CD55). Two of the Afa/Dr adhesions, AfaE-III and DraE, differ from each other by only three residues but are reported to have several different properties. One such difference is disruption of the interaction between DraE and CD55 by chloramphenicol, whereas binding of AfaE-III to CD55 is unaffected. Here we present a crystal structure of a strand-swapped trimer of wild type DraE. We also present a crystal structure of this trimer in complex with chloramphenicol, as well as NMR data supporting the binding position of chloramphenicol within the crystal. The crystal structure reveals the precise atomic basis for the sensitivity of DraE-CD55 binding to chloramphenicol and demonstrates that in contrast to other chloramphenicol-protein complexes, drug binding is mediated via recognition of the chlorine "tail" rather than via intercalation of the benzene rings into a hydrophobic pocket.