A signaling complex of Ca2+-calmodulin-dependent protein kinase IV and protein phosphatase 2A.

Stimulation of T lymphocytes results in a rapid increase in intracellular calcium concentration ([Ca2+]i) that parallels the activation of Ca2+-calmodulin-dependent protein kinase IV (CaMKIV), a nuclear enzyme that can phosphorylate and activate the cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). However, inactivation of CaMKIV occurs despite the sustained increase in [Ca2+]i that is required for T cell activation. A stable and stoichiometric complex of CaMKIV with protein serine-threonine phosphatase 2A (PP2A) was identified in which PP2A dephosphorylates CaMKIV and functions as a negative regulator of CaMKIV signaling. In Jurkat T cells, inhibition of PP2A activity by small t antigen enhanced activation of CREB-mediated transcription by CaMKIV. These findings reveal an intracellular signaling mechanism whereby a protein serine-threonine kinase (CaMKIV) is regulated by a tightly associated protein serine-threonine phosphatase (PP2A).

Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex.

Regulation of N-methyl-D-aspartate (NMDA) receptor activity by kinases and phosphatases contributes to the modulation of synaptic transmission. Targeting of these enzymes near the substrate is proposed to enhance phosphorylation-dependent modulation. Yotiao, an NMDA receptor-associated protein, bound the type I protein phosphatase (PP1) and the adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme. Anchored PP1 was active, limiting channel activity, whereas PKA activation overcame constitutive PP1 activity and conferred rapid enhancement of NMDA receptor currents. Hence, yotiao is a scaffold protein that physically attaches PP1 and PKA to NMDA receptors to regulate channel activity.

CT-based manual segmentation and evaluation of paranasal sinuses.

Manual segmentation of computed tomography (CT) datasets was performed for robot-assisted endoscope movement during functional endoscopic sinus surgery (FESS). Segmented 3D models are needed for the robots' workspace definition. A total of 50 preselected CT datasets were each segmented in 150-200 coronal slices with 24 landmarks being set. Three different colors for segmentation represent diverse risk areas. Extension and volumetric measurements were performed. Three-dimensional reconstruction was generated after segmentation. Manual segmentation took 8-10 h for each CT dataset. The mean volumes were: right maxillary sinus 17.4 cm(3), left side 17.9 cm(3), right frontal sinus 4.2 cm(3), left side 4.0 cm(3), total frontal sinuses 7.9 cm(3), sphenoid sinus right side 5.3 cm(3), left side 5.5 cm(3), total sphenoid sinus volume 11.2 cm(3). Our manually segmented 3D-models present the patient's individual anatomy with a special focus on structures in danger according to the diverse colored risk areas. For safe robot assistance, the high-accuracy models represent an average of the population for anatomical variations, extension and volumetric measurements. They can be used as a database for automatic model-based segmentation. None of the segmentation methods so far described provide risk segmentation. The robot's maximum distance to the segmented border can be adjusted according to the differently colored areas.

[Evaluation of force data with a force/torque sensor during FESS. A step towards robot-assisted surgery]. / Sensorbasierte Messung mechanischer Kräfte am Endoskop während FESS. Schritte zur robotergestützten Nasennebenhöhlenchirurgie.

BACKGROUND: To relieve the surgeon during functional endoscopic endonasal sinus surgery (FESS), the endoscope should be guided by autonomous robot assistance. The surgeon will thus have two hands free for suctioning and manipulation during FESS. PATIENTS/METHODS: With a force/torque sensor mounted on the endoscope, we measured forces in six degrees of freedom in five cadaver heads and in 20 actual endoscopic sinus procedures. On the cadaver heads we performed complete endoscopic endonasal dissection of all paranasal sinuses. All forces at the endoscope were monitored continuously. RESULTS: The mean forces occurring at the endoscope were 3.2 N. There were only slight differences between the in vivo and ex vivo data. We measured peak forces up to 25.2 N. In 95% of all cases, forces were lower than 7 N. CONCLUSION: Forces up to 7 N are sufficient for endoscopic guidance during FESS. Peak forces are distinctive for endoscopic guidance by humans and could be optimised by sensor-based intraoperative robot guidance. Higher forces are required for surgical endoscopy of the frontal and maxillary sinuses compared with the ethmoid sinuses.

Using robotic systems in order to determine biomechanical properties of soft tissues.

Biomechanical properties of soft tissue are important not only during computer simulation for medical training but also for systems where tissue deformation must be estimated in real-time, for example, Robot Assisted Surgery. The purpose of this paper is to describe some biomechanical tests consisting in the measurement of contact forces and deformations in tissue phantoms and porcine soft tissues (liver, brain, stomach and intestine). During the measurements two different procedures were applied. First, we have used a 5DOF micromanipulator instrumented with a spherical probe and a 6-axis force/torque ATI sensor. In the second procedure instead of the micromanipulator a Stäubli RX60 robot was used to apply the force over the samples. During this last test a high noise-signal relationship was detected and in order to improve the accuracy of the experiments some results were obtained using a Stäubli TX40 robot. Major accuracy in research in the field of soft tissue could be reached using standard procedures. Robotic systems allow precise movements to carry on biomechanical tests, and also permit a wide range of tasks to be implemented.

Robot-assisted fracture reduction: a preliminary study in the femur shaft.

Reduction in femoral shaft fractures can be difficult to achieve with minimally invasive techniques. Malalignment and high intra-operative radiation exposure can result. The hypothesis was that robot-assisted fracture reduction could improve the quality of reduction while reducing the amount of radiation exposure. A robot system was developed that allows fracture manipulation with a joystick as input device. The system provides the surgeon with haptic and metric feedback. Fifteen synthetic femurs were broken and reduced by simulated open (group A) and closed techniques (group B). These techniques were compared with the robot-assisted reduction with (group C) and without (group D) haptic and metric information. An image intensifier was simulated with two orthogonal cameras. All reduction techniques showed minor malalignment. In group C, the alignment was: procurvatum/recurvatum 0.6 degrees (0-2.0 degrees); varus/valgus 0.8 degrees (0-3.0 degrees); and axial rotation 0.8 degrees (0-3.1 degrees). A significant difference was seen between the groups (two-way ANOVA, p < 0.001). Axial rotation was significantly lower in group C than in group B (1.9 degrees; p < 0.001). The residual varus and valgus deviation was higher in group C compared with group A (0.4 degrees, p = 0.03). The median number of simulated radiographs was significantly less in group C (35) compared with group D (72; p < 0.001) and group B (49; p = 0.01). Robot-assisted fracture reduction of the femur provides high precision in alignment while reducing the amount of intraoperative imaging. Further research in this field is worthwhile.

Identification of rat serotonin 5-HT2C receptors as glycoproteins containing N-linked oligosaccharides.

Antibodies against a portion of the rat 5-HT2C receptor third intracellular loop were generated and used to identify receptors solubilized from cell lines and rat brain. Western blots of CHAPS-soluble proteins were probed with affinity-purified anti-2C antibodies. The specificity of anti-2C was demonstrated with extracts prepared from NIH/3T3 fibroblasts which stably express functional rat 5-HT2C or 5-HT2A receptors. Extracts from the 5-HT2C cell line, but not the 5-HT2A cell line, contained immunoreactive proteins with masses of 51-52 kDa and 58-68 kDa. In the brain, immunoreactive proteins were identified from choroid plexus extracts with masses of 51 kDa and 58-62 kDa. The major 58-62 kDa and minor 51 kDa proteins were not detected in extracts prepared from the hippocampus, striatum, or frontal cortex using the same amount of CHAPS-soluble protein. These results are consistent with previous studies demonstrating that 5-HT2C receptor binding sites and mRNA are most abundant in choroid plexus. The association of asparagine-linked (N-linked) oligosaccharides with the receptors was examined next. The 5-HT2C receptor cell line (3T3/2C) was grown in the presence of tunicamycin to metabolically inhibit N-linked glycosylation. Proteins from the cell extracts were detected with masses of 40 and 41 kDa. Extracts prepared from 3T3/2C cells (grown in the absence of tunicamycin) and from choroid plexus were incubated with N-glycosidase F to enzymatically remove available N-linked sugars. Immunoreactive proteins were detected with masses of 41 and 42 kDa from 3T3/2C cells and 41 kDa from choroid plexus. Neuraminidase, which cleaves sialic acid (N-acetylneuraminic acid) residues from glycoproteins, reduced the mass of the 51 and 58-62 kDa proteins from the choroid plexus to 50 and 54-58 kDa. In contrast, the 51-52 and 58-68 kDa proteins from 3T3/2C cells were not affected by treatment with neuraminidase. These results demonstrate that 5-HT2C receptors contain N-linked sugars and suggest that sialic acid residues associate with 5-HT2C receptors in the choroid plexus. The oligosaccharide moieties, which contribute up to approximately 30% of the relative mass as judged by SDS-polyacrylamide gel electrophoresis, may impart functional properties to 5-HT2C receptors.

Protein serine/threonine phosphatase 1 and 2A associate with and dephosphorylate neurofilaments.

The phosphorylation state of neurofilaments plays an important role in the control of cytoskeletal integrity, axonal transport, and axon diameter. Immunocytochemical analyses of spinal cord revealed axonal localization of all protein phosphatase subunits. To determine whether protein phosphatases associate with axonal neurofilaments, neurofilament proteins were isolated from bovine spinal cord white matter by gel filtration. approximately 15% of the total phosphorylase a phosphatase activity was present in the neurofilament fraction. The catalytic subunits of PP1 and PP2A, as well as the A and B alpha regulatory subunits of PP2A, were detected in the neurofilament fraction by immunoblotting, whereas PP2B and PP2C were found exclusively in the low molecular weight soluble fractions. PP1 and PP2A subunits could be partially dissociated from neurofilaments by high salt but not by phosphatase inhibitors, indicating that the interaction does not involve the catalytic site. In both neurofilament and soluble fractions, 75% of the phosphatase activity towards exogenous phosphorylase a could be attributed to PP2A, and the remainder to PP1 as shown with specific inhibitors. Neurofilament proteins were phosphorylated in vitro by associated protein kinases which appeared to include protein kinase A, calcium/calmodulin-dependent protein kinase, and heparin-sensitive and -insensitive cofactor-independent kinases. Dephosphorylation of phosphorylated neurofilament subunits was mainly (60%) catalyzed by associated PP2A, with PP1 contributing minor activity (10-20%). These studies suggest that neurofilament-associated PP1 and PP2A play an important role in the regulation of neurofilament phosphorylation.

Clinical experience with stentless pericardial aortic monopatch for aortic valve replacement.

A stentless pericardial aortic monopatch was used in 60 consecutive patients undergoing aortic valve replacement. The monopatch is constructed of a sheet of glutaraldehyde-treated bovine pericardium, tailored and shaped to fit the aortic anulus, and is sutured in place without a stent or sewing ring. The valve area is effectively preserved by this method. Results indicate that this technique is simple, inexpensive, and applicable to all cases of aortic valve disease. It does not require anticoagulation and may allow for annular growth when used in children. This technique is particularly suitable for patients with infective endocarditis because the amount of foreign material is minimized.

Antileukocyte antibodies in patients refractory to platelet transfusions.

Sera from 12 multitransfused patients who were refractory to random-donor platelets were tested for lymphocytotoxic and leukoagglutinating antibodies using panel cells from various volunteers whose HLA-A and -B antigens were known. All sera contained leukoagglutinins reactive with cells from at least one panel member, whereas only 33% had lymphocytotoxic antibodies. Patients whose sera reacted frequently with panel cells using the microcapillary agglutination technic seldom responded to HLA-matched paltelets, whereas those whose sera reacted infrequently usually responded satisfactorily. It is concluded that non-HLA antibodies may play a significant role in determining the responses to platelet transfusions in multi-transfused patients.

Rapid thawing of fresh frozen plasma.

Fresh frozen plasma (FFP) normally requires about 45 min to thaw in a 37 degrees C water bath when placed inside an additional plastic overbag. That relatively prolonged time may result in non-utilization or delays in delivery of the product, especially, during emergency surgery. One report recommends the use of a microwave oven to overcome those problems. Most blood banks do not have microwave ovens but usually do have water baths at 56 degrees C. Ten units of FFP thawed inside plastic overbags at 37 degrees C required 49.5 +/- 3.9 min and 22 units thawed at 56 degrees C required 28.3 +/- 4 min to thaw. Removal of the plastic overbag reduces the thawing time to 12-13 min at 56 degrees C. The activity Factor V was 85 +/- 15% (65-106%) at 37 degrees C and 80 +/- 21% (47-118%) at 56 degrees C. Factor VIII activity was 86 +/- 21% (59-118%) at 37 degrees C and 90 +/- 37% (46-225%) at 56 degrees C. There were no demonstrable alterations in fibrinogen, PT, APTT, Factor II, VII, IX, and XI between the two thawing temperatures, even after 24 hours of storage at 4 degrees C.

The safety of blood components and derivatives.

An overview of the steps taken to ensure the safety of blood components and derivatives is provided. A brief discussion of hereditary bleeding disorders (hemophilia A, B, and von Willebrand's disease) is included, and the safety of derivatives available for their treatment is discussed, as well as the method of production and the level of safety of derivatives such as factor VIII and factor IX concentrates.

Optimization of antibody fragment production in Bacillus megaterium: the role of metal ions on protein secretion.

The effect of the concentration of metal ions in minimal media has been shown to be very important for the production and secretion of the antibody fragment D1.3 scFv in Bacillus megaterium YYBm1. The best media compositions for biomass and antibody fragment formation were evaluated using a genetic algorithm. The screening was carried out in 96 microtiter deep well plates with 900 µL cultivation volume. In 7 generations, 240 different kinds of media were tested, key elements for production and secretion were detected and a 117% increase in production of antibody fragment compared to the previously used medium could be achieved. In addition, media with a higher biomass formation (+84%) or with both more biomass and a higher production of antibody fragment (Pareto-front members) were found. Interestingly the best media for protein production and secretion were different in their composition, with regards to the metal ion concentration levels. From data derived experimentally and from the genome, magnesium was shown to be one of the key components of the metal ions tested for biomass formation and especially for production and secretion of the antibody fragment D1.3 scFv.

Development of a fixation device for robot assisted fracture reduction of femoral shaft fractures: a biomechanical study.

Robot assisted fracture reduction of femoral shaft fractures provides precise alignment while reducing the amount of intraoperative imaging. The connection between the robot and the fracture fragment should allow conventional intramedullary nailing, be minimally invasive and provide interim fracture stability. In our study we tested three different reduction tools: a conventional External Fixator, a Reposition-Plate and a Three-Point-Device with two variations (a 40 degrees and a 90 degrees version). We measured relative movements between the tools and the bone fragments in all translation and rotation planes. The Three-Point-Device 90 degrees showed the smallest average relative displacement and was the only device able to withstand the maximum applied load of 70 Nm without failure of any bone fragment. The Three-Point-Device 90 degrees complies with all the stipulated requirements and is a suitable interface for robot assisted fracture reduction of femoral shaft fractures.