High-resolution atomic force microscopy imaging of rhodopsin in rod outer segment disk membranes.

Atomic force microscopy (AFM) is a powerful imaging technique that allows recording topographical information of membrane proteins under near-physiological conditions. Remarkable results have been obtained on membrane proteins that were reconstituted into lipid bilayers. High-resolution AFM imaging of native disk membranes from vertebrate rod outer segments has unveiled the higher-order oligomeric state of the G protein-coupled receptor rhodopsin, which is highly expressed in disk membranes. Based on AFM imaging, it has been demonstrated that rhodopsin assembles in rows of dimers and paracrystals and that the rhodopsin dimer is the fundamental building block of higher-order structures.

Reduced trabecular bone mineral density and cortical thickness accompanied by increased outer bone circumference in metacarpal bone of rheumatoid arthritis patients: a cross-sectional study

Introduction The objective of this study was to assess three-dimensional bone geometry and density at the epiphysis and shaft of the third meta-carpal bone of rheumatoid arthritis (RA) patients in comparison to healthy controls with the novel method of peripheral quantitative computed tomography (pQCT). Methods PQCT scans were performed in 50 female RA patients and 100 healthy female controls at the distal epiphyses and shafts of the third metacarpal bone, the radius and the tibia. Reproducibility was determined by coefficient of varia-tion. Bone densitometric and geometric parameters were compared between the two groups and correlated to disease characteristics. Results Reproducibility of different pQCT parameters was between 0.7% and 2.5%. RA patients had 12% to 19% lower trabecular bone mineral density (BMD) (P ≤ 0.001) at the distal epiphyses of radius, tibia and metacarpal bone. At the shafts of these bones RA patients had 7% to 16% thinner cortices (P ≤ 0.03). Total cross-sectional area (CSA) at the metacarpal bone shaft of pa-tients was larger (between 5% and 7%, P < 0.02), and relative cortical area was reduced by 13%. Erosiveness by Ratingen score correlated negatively with tra-becular and total BMD at the epiphyses and shaft cortical thickness of all measured bones (P < 0.04). Conclusions Reduced trabecular BMD and thinner cortices at peripheral bones, and a greater bone shaft diameter at the metacarpal bone suggest RA spe-cific bone alterations. The proposed pQCT protocol is reliable and allows measuring juxta-articular trabecular BMD and shaft geometry at the metacarpal bone.

Outer skull landmark-based coordinates for measurement of cerebral blood flow and intracranial pressure in rabbits

Despite the increased use of intracranial neuromonitoring during experimental subarachnoid hemorrhage (SAH), coordinates for probe placement in rabbits are lacking. This study evaluates the safety and reliability of using outer skull landmarks to identify locations for placement of cerebral blood flow (CBF) and intraparenchymal intracranial pressure (ICP) probes. Experimental SAH was performed in 17 rabbits using an extracranial-intracranial shunt model. ICP probes were placed in the frontal lobe and compared to measurements recorded from the olfactory bulb. CBF probes were placed in various locations in the frontal cortex anterior to the coronary suture. Insertion depth, relation to the ventricular system, and ideal placement location were determined by post-mortem examination. ICP recordings at the time of SAH from the frontal lobe did not differ significantly from those obtained from the right olfactory bulb. Ideal coordinates for intraparenchymal CBF probes in the left and right frontal lobe were found to be located 4.6±0.9 and 4.5±1.2 anterior to the bregma, 4.7±0.7mm and 4.7±0.5mm parasagittal, and at depths of 4±0.5mm and 3.9±0.5mm, respectively. The results demonstrate that the presented coordinates based on skull landmarks allow reliable placement of intraparenchymal ICP and CBF probes in rabbit brains without the use of a stereotactic frame.

The copper-inducible ComR (YcfQ) repressor regulates expression of ComC (YcfR), which affects copper permeability of the outer membrane of Escherichia coli

The pathway of copper entry into Escherichia coli is still unknown. In an attempt to shed light on this process, a lux-based biosensor was utilized to monitor intracellular copper levels in situ. From a transposon-mutagenized library, strains were selected in which copper entry into cells was reduced, apparent as clones with reduced luminescence when grown in the presence of copper (low-glowers). One low-glower had a transposon insertion in the comR gene, which encodes a TetR-like transcriptional regulator. The mutant strain could be complemented by the comR gene on a plasmid, restoring luminescence to wild-type levels. ComR did not regulate its own expression, but was required for copper-induction of the neighboring, divergently transcribed comC gene, as shown by real-time quantitative PCR and with a promoter-lux fusion. The purified ComR regulator bound to the promoter region of the comC gene in vitro and was released by copper. By membrane fractionation, ComC was shown to be localized in the outer membrane. When grown in the presence of copper, ∆comC cells had higher periplasmic and cytoplasmic copper levels, compared to the wild-type, as assessed by the activation of the periplasmic CusRS sensor and the cytoplasmic CueR sensor, respectively. Thus, ComC is an outer membrane protein which lowers the permeability of the outer membrane to copper. The expression of ComC is controlled by ComR, a novel, TetR-like copper-responsive repressor.

High-resolution imaging of 2D outer membrane protein F (OmpF) crystals by atomic force microscopy

In this chapter the methodological bases are provided to achieve subnanometer resolution on two-dimensional (2D) membrane protein crystals by atomic force microscopy (AFM). This is outlined in detail with the example of AFM studies of the outer membrane protein F (OmpF) from the bacterium Escherichia coli (E. coli). We describe in detail the high-resolution imaging of 2D OmpF crystals in aqueous solution and under near-physiological conditions. The topographs of OmpF, and stylus effects and artifacts encountered when imaging by AFM are discussed.

Influence of Durotomy on Laser-Doppler Measurement of Spinal Cord Blood Flow in Chondrodystrophic Dogs with Thoracolumbar Disk Extrusion

OBJECTIVES: To assess influence of durotomy on spinal cord blood flow (SCBF) in chondrodystrophic dogs with thoracolumbar disk extrusion. STUDY DESIGN: Prospective cohort study. ANIMALS: Chondrodystrophic dogs with thoracolumbar disk extrusion (n = 11). METHODS: Diagnosis was based on neurologic signs, magnetic resonance imaging (MRI) findings, and surgical confirmation. Regional SCBF was measured 3 times intraoperatively by laser-Doppler flowmetry: (1) before surgical decompression; (2) immediately after decompression by hemilaminectomy-durotomy; and (3) after 15 minutes of lesion lavage. A standardized hemilaminectomy and durotomy performed by the same neurosurgeon, was used to minimize factors that could influence measurement readings. RESULTS: A significant increase in intraoperative SCBF was found immediately after spinal cord decompression and durotomy in dogs but SCBF returned to previous levels or lower after 15 minutes of lavage. Changes in SCBF were not associated with duration of clinical signs; neurologic status, degree of spinal cord compression, or signal intensity changes as assessed by MRI. CONCLUSION: Durotomy does not increase SCBF in dogs with disk extrusion associated spinal cord compression.

Microfenestration using the CUSA Excel ultrasonic aspiration system in chondrodystrophic dogs with thoracolumbar disk extrusion: a descriptive cadaveric and clinical study

OBJECTIVE: To describe an ultrasonic surgical aspirator assisted disk fenestration technique in dogs. STUDY DESIGN: Descriptive cadaveric and prospective clinical study. ANIMALS: Fresh Beagle cadavers (n=5) and 10 chondrodystrophic dogs with thoracolumbar disk extrusion. METHODS: Cadaveric study: Intervertebral disks T12-L2 were fenestrated with the CUSA Excel in 5 Beagle cadavers, and fenestration efficacy assessed by morphologic examination of the completeness of fenestration and size of annulotomy. Clinical study: the affected intervertebral disk was fenestrated in 10 chondrodystrophic dogs treated by hemilaminectomy for thoracolumbar disk disease. Efficacy of fenestration was evaluated. RESULTS: Mean time necessary to perform CUSA assisted fenestration was 8 minutes (range, 5-10 minutes) for each disk in cadavers and patients. In cadaver spines, removal of the nucleus pulposus was complete in 11/15 disks. In 4 disks, remnants of nucleus pulposus material were observed on the contralateral side. Nuclear material was normal in 9/15 disks and showed evidence of chondroid degeneration on histopathologic examination in the 6 disks. Median annulotomy size was 3 mm. Clinically, no signs of early recurrence were observed and all dogs recovered uneventfully. CONCLUSIONS: CUSA assisted fenestration is a safe and efficient method of fenestration for removal of most of the nucleus pulposus through a limited annulotomy.

Intraobserver and interobserver agreement for results of low-field magnetic resonance imaging in dogs with and without clinical signs of disk-associated wobbler syndrome

OBJECTIVE: To determine interobserver and intraobserver agreement for results of low-field magnetic resonance imaging (MRI) in dogs with and without disk-associated wobbler syndrome (DAWS). DESIGN: Validation study. ANIMALS: 21 dogs with and 23 dogs without clinical signs of DAWS. PROCEDURES: For each dog, MRI of the cervical vertebral column was performed. The MRI studies were presented in a randomized sequence to 4 board-certified radiologists blinded to clinical status. Observers assessed degree of disk degeneration, disk-associated and dorsal compression, alterations in intraspinal signal intensity (ISI), vertebral body abnormalities, and new bone formation and categorized each study as originating from a clinically affected or clinically normal dog. Interobserver agreement was calculated for 44 initial measurements for each observer. Intraobserver agreement was calculated for 11 replicate measurements for each observer. RESULTS: There was good interobserver agreement for ratings of disk degeneration and vertebral body abnormalities and moderate interobserver agreement for ratings of disk-associated compression, dorsal compression, alterations in ISI, new bone formation, and suspected clinical status. There was very good intraobserver agreement for ratings of disk degeneration, disk-associated compression, alterations in ISI, vertebral body abnormalities, and suspected clinical status. There was good intraobserver agreement for ratings of dorsal compression and new bone formation. Two of 21 clinically affected dogs were erroneously categorized as clinically normal, and 4 of 23 clinically normal dogs were erroneously categorized as clinically affected. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that variability exists among observers with regard to results of MRI in dogs with DAWS and that MRI could lead to false-positive and false-negative assessments.

Suspected paradoxical nerve root signature in a dog with right-sided intervertebral lumbar disk extrusion: a case report

A 7 year old male mongrel dog was presented with a 3 weeks history of gait disturbance in the pelvic limbs more pronounced on the left side associated with pain in the lumbar spine. At presentation neurologic deficits consisted of mild bilateral proprioceptive deficits and nerve root signature in the left pelvic limb. A large intervertebral disc herniation L3-L4 located in a right ventrolateral area of the spinal canal was diagnosed by magnetic resonance imaging. The herniated disc was removed through right hemilaminectomy and fenestration. The dog recovered quickly and returned to the owners 4 days after surgery with a slight lameness in the left pelvic limb. On the follow-up examination 2 months later the dog showed normal gait and normal neurological examination. Nerve root signature is not always indicative for the side of the lesion in case of lateralized intervertebral disc herniation