Polo-like kinase is required for the fragmentation of pericentriolar Golgi stacks during mitosis.

The pericentriolar stacks of Golgi cisternae undergo extensive reorganization during mitosis in mammalian cells. GM130 and GRASP65 (Golgi reassembly stacking protein of 65 kDa) are Golgi-associated proteins that are targets of mitotic kinases, and they have also been implicated in the reorganization of the Golgi structure during cell division. Previous studies have reported that mitogen-activated protein kinase kinase-1 (MEK1) and Cdc2 protein kinases are involved in these dynamic changes in the Golgi structure. More recently, the mitotic polo-like kinase (Plk) has been shown to interact with and phosphorylate GRASP65. Here, we provide evidence that Plk is involved in the mitosis-specific fragmentation of the Golgi apparatus. The addition of kinase-defective Plk or immunodepletion of Plk disrupts the fragmentation process. Furthermore, Golgi fragmentation is inhibited by the addition of either full-length or truncated GRASP65. These findings suggest that phosphorylation of GRASP65 by Plk may be a critical event in the reorganization of the Golgi structure during mitosis.

Biomechanical evaluation of occipitocervical fixation devices.

Human cadaveric occipitocervical specimens were implanted with three types of instrumentation. The devices were tested biomechanically under three modes of loading to determine the stiffness of spinal constructs and the failure mechanisms of the constructs under extreme flexion. The devices tested were the AXIS Fixation System (with custom plate), the Y-Plate, and the Luque rectangle. No significant differences in stiffness among the devices were found under compression and flexion. The stiffnesses of the plate systems were statistically higher than the Luque rectangle in extension and torsion. In extreme flexion, the plate systems failed by fracture of the C2 pedicles. Modern plate systems, for occipitocervical fixation, provide more stiffness and stability than traditional wiring techniques. This study provides surgeons with information on the relative merits of modern plate and screw systems compared with traditional rod and wire constructs.

Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei.

Trypanosoma brucei, the protozoan parasite responsible for sleeping sickness, evades the immune response of mammalian hosts and digestion in the gut of the insect vector by means of its coat proteins tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. To evaluate the importance of GPI for parasite survival, we cloned and disrupted a trypanosomal gene, TbGPI10, involved in biosynthesis of GPI. TbGPI10 encodes a protein of 558 amino acids having 25% and 23% sequence identity to human PIG-B and Saccharomyces cerevisiae Gpi10p, respectively. TbGPI10 restored biosynthesis of GPI in a mouse mutant cell line defective in mouse Pig-b gene. TbGPI10 also rescued the inviability of GPI10-disrupted S. cerevisiae, indicating that TbGPI10 is the orthologue of PIG-B/GPI10 that is involved in the transfer of the third mannose to GPI. The bloodstream form of T. brucei could not lose TbGPI10; therefore, GPI synthesis is essential for growth of mammalian stage parasites. Procyclic form cells (insect stage parasites) lacking the surface coat proteins because of disruption of TbGPI10 are viable and grow slower than normal, provided that they are cultured in nonadherent flasks. In regular flasks, they adhered to the plastic surface and died. Infectivity to tsetse flies is partially impaired, particularly in the early stage. Therefore, parasitespecific inhibition of GPI biosynthesis should be an effective chemotherapy target against African trypanosomiasis.

Sphingoid base synthesis requirement for endocytosis in Saccharomyces cerevisiae.

The internalization step of endocytosis in yeast requires actin and sterols for maximum efficiency. In addition, many receptors and plasma membrane proteins must be phosphorylated and ubiquitylated prior to internalization. The Saccharomyces cerevisiae end8-1 mutant is allelic to lcb1, a mutant defective in the first step of sphingoid base synthesis. Upon arrest of sphingoid base synthesis a rapid block in endocytosis is seen. This block can be overcome by exogenous sphingoid base. Under conditions where endogenous sphingosine base synthesis was blocked and exogenous sphingoid bases could not be converted to phosphorylated sphingoid bases or to ceramide, sphingoid bases could still suppress the endocytic defect. Therefore, the required lipid is most likely a sphingoid base. Interestingly, sphingoid base synthesis is required for proper actin organization, but is not required for receptor phosphorylation. This is the first case of a physiological role for sphingoid base synthesis, other than as a precursor for ceramide or phosphorylated sphingoid base synthesis.

A nucleoside transporter from Trypanosoma brucei involved in drug resistance.

Drug resistance of pathogens is an increasing problem whose underlying mechanisms are not fully understood. Cellular uptake of the major drugs against Trypanosoma brucei spp., the causative agents of sleeping sickness, is thought to occur through an unusual, so far unidentified adenosine transporter. Saccharomyces cerevisiae was used in a functional screen to clone a gene (TbAT1) from Trypanosoma brucei brucei that encodes a nucleoside transporter. When expressed in yeast, TbAT1 enabled adenosine uptake and conferred susceptibility to melaminophenyl arsenicals. Drug-resistant trypanosomes harbor a defective TbAT1 variant. The molecular identification of the entry route of trypanocides opens the way to approaches for diagnosis and treatment of drug-resistant sleeping sickness.

Sweet's syndrome associated with spinal surgical intervention. A case report.

STUDY DESIGN: A patient with a medical history of Sweet's Syndrome, an acute neutrophilic dermatosis, was seen at the authors' institution for cervical pain. After undergoing a thorough history-taking and physical examination and after experiencing no relief with conservative therapy, the patient underwent cervical spine surgery. After the surgical procedure, the patient developed multiple cutaneous lesions that were consistent with the findings associated with an acute recurrence of Sweet's Syndrome. OBJECTIVES: To characterize the authors' experience with this unusual histologically documented dermatologic disorder. SUMMARY OF BACKGROUND DATA: Sweet's Syndrome is a rare form of neutrophilic dermatosis characterized by recurrent eruptions of painful, edematous, red, tender plaques that are found predominantly on the torso in middle-aged women. After an extensive literature search, it was noted that this rare and unusual disorder has not been reported previously in association with surgical intervention of any type, including spinal operations. METHODS: The patient's postoperative course was documented, and all medical records were reviewed retrospectively. RESULTS: The patient's rash resolved spontaneously. Solid fusion of C5-C6 occurred. The patient remained neurovascularly intact, and her axial cervical pain decreased significantly from its preoperative levels. CONCLUSIONS: Sweet's Syndrome remains a rare dermatologic disorder, which may complicate a routine postoperative course. Patients with Sweet's Syndrome have an exceedingly high rate of other serious medical illness. The effect of Sweet's Syndrome on physiologic bone healing is unknown. In this patient, there was nonunion of the cervical spine, with eventual solid bony union. Perioperatively, patients with this disorder are treated with oral prednisone and oral antibiotics to prevent secondary complications at the surgical wound.

Saccharomyces cerevisiae GPI10, the functional homologue of human PIG-B, is required for glycosylphosphatidylinositol-anchor synthesis.

An increasing number of plasma membrane proteins have been shown to be attached to the membrane via a glycosylphosphatidylinositol (GPI) moiety. All eukaryotes share a highly conserved GPI-core structure EthN-P-Man3-GlcN-PI, where EthN is ethanolamine. We have identified a protein encoded by the yeast open reading frame YGL142C that shares 33% identity with the human Pig-B protein. Deletion of this essential gene leads to a block in GPI anchor biosynthesis. We therefore named the gene GPI10. Gpi10p and Pig-B are functional homologues and the lethal deletion of GPI10 can be rescued by expression of the PIG-B cDNA. As found for PIG-B mutant cells, gpi10 deletant cells cannot attach the third mannose in an alpha-1,2 linkage to the GPI core-structure intermediate. Overexpression of GPI10 gives partial resistance to the GPI-synthesis inhibitor YW3548, suggesting that this gene product may affect the target of the inhibitor.

Identification of a species-specific inhibitor of glycosylphosphatidylinositol synthesis.

Glycosylphosphatidylinositol (GPI)-anchoring represents a mechanism for attaching proteins to the cell surface that is used among all eukaryotes. A common core structure, EthN-P-Man3-GlcN-PI, is synthesized by sequential transfer of sugars and ethanolamine-P to PI and is highly conserved between organisms. We have screened for natural compounds that inhibit GPI-anchoring in yeast and have identified a terpenoid lactone, YW3548, that specifically blocks the addition of the third mannose to the intermediate structure Man2-GlcN-acyIPI. Consistent with the block in GPI synthesis, YW3548 prevents the incorporation of [3H]myo-inositol into proteins, transport of GPI-anchored proteins to the Golgi and is toxic. The compound inhibits the same step of GPI synthesis in mammalian cells, but has no significant activity in protozoa. These results suggest that despite the conserved core structure, the GPI biosynthetic machinery may be different enough between mammalian and protozoa to represent a target for anti-protozoan chemotherapy.

Rigid internal fixation with lateral mass plates in multilevel anterior and posterior reconstruction of the cervical spine.

STUDY DESIGN: A retrospective clinical and radiographic analysis was performed on 17 patients with multilevel cervical disease who were treated with anterior and posterior reconstruction with a new rigid, segmental, internal fixation system applied to the lateral masses. OBJECTIVES: To determine the applicability, safety, and clinical efficacy of an instrumentation system used as a cervical lateral mass plate in the management of complex spinal disorders. SUMMARY OF BACKGROUND DATA: Cervical disorders involving three or more levels present a difficult reconstruction problem, especially if the posterior elements are deficient. Segmental fixation with lateral mass plating provides an alternative method to situations that would otherwise require a halo. METHODS: Seventeen patients treated by a single surgeon underwent cervical reconstruction surgery involving three or more levels. All patients had anterior decompression and reconstruction and a posterior fusion with rigid internal fixation with a device applied to the lateral masses of the cervical vertebrae. Patients were reviewed clinically and radiographically to determine the efficacy and safety of this method of fixation. RESULTS: Of the 15 patients with adequate follow-up data that were studied, the condition of 13 patients, (87%) was improved, that of one patient (6.7%) was the same, and that of another (6.7%) was worse after surgical intervention. Sagittal alignment was restored to within 5 degrees of the preoperative lordosis in active extension by the modified Cobb method and the Gore method. No patient had radiographic nonunion. One patient had a sensory radiculopathy associated with an overpenetrated lateral mass screw that partially resolved after hardware removal. One patient had asymptomatic loosening of a C7 lateral mass screw. CONCLUSIONS: Segmental posterior fixation with lateral mass plating provides more rigid immobilization than traditional techniques, allows restoration and maintenance of spinal alignment, obviates the need for halo immobilization, and is associated with a low incidence of neurovascular injury.

Specific requirements for the ER to Golgi transport of GPI-anchored proteins in yeast.

GPI-anchored proteins are attached to the membrane via a glycosylphosphatidylinositol-(GPI) anchor whose carbohydrate core is conserved in all eukaryotes. Apart from membrane attachment, the precise role of the GPI-anchor is not known, but it has been proposed to play a role in protein sorting. We have investigated the transport of the yeast GPI-anchored protein Gas1p. We identified two mutant strains involved in very different cellular processes that are blocked selectively in the transport of GPI-anchored proteins before arrival to the Golgi. The end8-1/lcb1-100 mutant is defective in ceramide synthesis. In vitro data suggest a requirement for ceramides after the exit from the ER. We therefore propose that ceramides might function in the fusion of a GPI-containing vesicle with the Golgi, but we cannot exclude a role in the ER. The second mutant that blocks the transport of GPI-anchored proteins to the Golgi is ret1-1, a mutant in the alpha-subunit of coatomer. In both mutants, GPI-anchor attachment is normal and in ret1-1 cells, the GPI-anchors are remodeled with ceramide to the same extent as in wild-type cells.

Reconstructive spinal surgery: assessment of outcome.

One of the foremost problems in evaluating patients who have had spinal surgery is the difficulty in reliably assessing changes in symptoms and function attributable to the operative procedure. In this study, a questionnaire was sent to patients who had had spinal surgery at least 6 months earlier. The data were entered into a data base that contained information about the surgical procedure. Correlation studies were conducted and t tests were used to find statistically significant differences. Seventy-two patients returned the completed questionnaire. The rate of return to work was significantly lower among those involved in workers' compensation or litigation and those with prolonged preoperative unemployment. Depression inversely correlated with satisfaction, the number of dissatisfied patients correlating directly with the number of patients significantly depressed. Physical deconditioning, change in attitude and perception, preinjury job dissatisfaction, secondary gain, and other medical conditions significantly decreased the probabilities of return to work and satisfaction.

The efficacy of pedicle screw/plate fixation on lumbar/lumbosacral autogenous bone graft fusion in adult patients with degenerative spondylolisthesis.

A total of 18 patients with grade I or II degenerative spondylolisthesis fused three levels or fewer with autogenous bone graft were entered at three clinical sites. After 2 years, these patients were found to have a fusion rate of 89%. A statistical analysis of these results compared with those in the literature showed that patients with spondylolisthesis who underwent fusion with pedicle screw instrumentation were > 3 times more likely to fuse than comparable patients implanted without a pedicle screw/plate system. The pedicle screw/plate system used in this study was shown to be an effective method of facilitating lumbar or lumbosacral fusion with autogenous bone graft for adult patients with a primary indication of grade I or II degenerative spondylolisthesis.

Complications of posterior cervical plating.

STUDY DESIGN: This retrospective clinical study evaluates complications occurring during or immediately after surgery of posterior cervical plating. OBJECTIVES: The present study quantifies risks associated with posterior cervical plating using lateral mass screw fixation. The observed clinical complications are compared with theoretical risks previously studied in cadavers. Unanticipated complications are identified. SUMMARY OF BACKGROUND DATA: There are many reports that describe posterior cervical plating and attempt to describe the indications for using this type of fixation, but few studies have discussed the clinical complications incurred by application of these plates and screws. METHODS: Seventy-eight consecutive patients whose treatment included posterior cervical lateral mass plating were independently reviewed to identify associated complications. The average patient age was 52.9 years, and the average follow-up period was 2 years (range, 10-47 months). Multiple indications for surgery were present, but complex reconstructive procedures were required in 70.5% of cases. Complication rates were calculated as either a percentage of the number of screws inserted or as a percentage of the number of cases performed or both. RESULTS: Six hundred fifty-four screws were inserted--an average of 8.4 screws per patient. Complication rates as a function of the number of screws inserted included nerve root injury, 0.6%; facet violations, 0.2%; vertebral artery injury, 0%; broken screw, 0.3%; screw avulsion, 0.2%; and screw loosening 1.1%. Complications as a percentage of the number of cases performed included spinal cord injury, 2.6%; iatrogenic foraminal stenosis, 2.6%; broken plate, 1.3%; lost reduction, 2.6%; adjacent segment degeneration, 3.8%; infection, 1.3%; and pseudoarthrosis, 1.4%. CONCLUSIONS: Cadaveric work has predicted certain anatomic complication rates associated with lateral mass screw insertion. This study finds the risk of lateral mass screw insertion to be considerably less than predicted in vitro. The present study reports other complications that were not predicted in laboratory studies.

The effect of pedicle screw/plate fixation on lumbar/lumbosacral autogenous bone graft fusions in patients with degenerative disc disease.

STUDY DESIGN: A prospective, multi-center Investigational Device Exemption Study was carried out in the United States using a pedicle screw and plate system to perform a fusion in patients with degenerative disc disease or spondylolisthesis. The patients' pain function, complications, and fusion status were evaluated and compared with literature controls. OBJECTIVES: To study the safety and efficacy of the ISF pedicle screw/plate system. This article focuses only on those study patients with degenerative disc disease treated with autogenous bone grafts and compares the results to those of similar patients treated without instrumentation, as reported in the literature. SUMMARY OF BACKGROUND DATA: Twenty-eight patients were in the subgroup studied--patients with degenerative disc disease who had fusions with autogenous bone graft. This study was conducted at four clinical sites with a 2-year follow-up. Patient follow-up was greater than 95% at all time points. METHODS: To be considered a patient with degenerative disc disease, radiographs had to demonstrate a collapse of the disc, the presence of bone erosion, or the compression of the vertebrae as the primary spinal abnormality. Spinal fusion must have been the recommended surgical treatment for discogenic pain. The fusion status was evaluated by the operating surgeon and an independent reviewer. RESULTS: After 2 years, this subset of patients (n = 28) with degenerative disc disease who had lumbar/lumbosacral fusion with autogenous bone graft was found to have a pseudarthrosis rate of 0%. Eight articles in the literature were found to be valid noninstrumented literature controls with which this subgroup could be compared. The average pseudarthrosis rate in the control group was 32%. CONCLUSIONS: A statistical analysis showed that patients with degenerative disc disease who underwent fusion without pedicle screw instrumentation were over 24 times more likely to have a pseudarthrosis than comparable patients implanted with a pedicle screw/plate system. Regarding the most important goal in performing a spinal fusion--fusion of the spine--the pedicle screw/plate system used in this study was shown to be a safe and efficacious method of facilitating fusion with autogenous bone graft for this patient population.

Gorham disease of the spine.

STUDY DESIGN: This is a case report of Gorham disease of the spine with review of the literature. OBJECTIVE: To review the diagnosis, therapy, clinical course, and prognosis of Gorham disease of the spine. SUMMARY OF BACKGROUND DATA: This is the 17th reported case of spinal involvement by this rare type of idiopathic osteolysis. METHODS: The patient was studied with radiographs, nuclear bone scans, computed tomography scans, magnetic resonance scans, and biopsies. His spine was stabilized by posterior and anterior rods, corpectomies, and bone grafts. RESULTS: The patient's spine had remained stable for 22 months after surgery, but intractable chylothoraces and spreading bone destruction were present. CONCLUSIONS: Spinal Gorham disease has high morbidity and mortality, but the course in an individual patient is difficult to predict. Early spinal stabilization should be considered before irreversible neurologic complications occur.

Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast.

Inhibition of ceramide synthesis by a fungal metabolite, myriocin, leads to a rapid and specific reduction in the rate of transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus without affecting transport of soluble or transmembrane proteins. Inhibition of ceramide biosynthesis also quickly blocks remodelling of GPI anchors to their ceramide-containing, mild base-resistant forms. These results suggest that the pool of ceramide is rapidly depleted from early points of the secretory pathway and that its presence at these locations enhances transport of GPI-anchored proteins specifically. A mutant that is resistant to myriocin reverses its effect on GPI-anchored protein transport without reversing its effects on ceramide synthesis and remodelling. Two hypotheses are proposed to explain the role of ceramide in the transport of GPI-anchored proteins.

Cell cycle-dependent expression of Nek2, a novel human protein kinase related to the NIMA mitotic regulator of Aspergillus nidulans.

The serine/threonine protein kinase NIMA of Aspergillus nidulans is required for entry into mitosis and may function in parallel to the universal mitotic inducer p34cdc2. Here, we report the isolation of complementary DNAs encoding Nek2 and Nek3, two novel human protein kinases structurally related to NIMA. Sequence comparisons revealed several unique features which may define a family of NIMA-related protein kinases. Nek2 was chosen for further study since it represents the closest known mammalian relative of NIMA. Chromosomal mapping of the nek2 gene identified two independent loci on chromosomes 1 and 14, and Northern blot analyses revealed the expression of two distinct mRNAs of approximately 2.4 and 4.7 kilobases in all human cell lines examined. In HeLa cells synchronized by both drug arrest and elutriation, a strikingly cell cycle-dependent pattern of Nek2 expression could be observed; Nek2 protein was almost undetectable during G1 but accumulated progressively throughout S, reaching maximal levels in late G2. These observations demonstrate that Nek2 resembles Aspergillus NIMA, not only in its catalytic domain, but also in its cell cycle-dependent expression. Hence, the human Nek2 protein kinase may also function at the onset of mitosis.

The effect of spinal implant rigidity on vertebral bone density. A canine model.

An animal model of anterior and posterior column instability was developed to allow in vivo observation of bone remodeling and arthrodesis following spinal instrumentation. After an initial anterior and posterior destabilizing lesion was created at the L5-L6 vertebral levels in 63 adult beagles, various spinal reconstructive surgical procedures were performed--with or without bilateral posterolateral bone grafting, with or without bilateral oophorectomies, and with or without spinal instrumentation (Harrington distraction, Luque rectangular, Cotrel-Dubousset pedicular, or Steffee pedicular implants). Observation 6 months after surgery revealed a significantly improved probability of achieving a spinal fusion if spinal instrumentation had been used (X2 = 5.84, P = .016). Nondestructive mechanical testing after removal of all metal instrumentation in torsion, axial compression, and flexion revealed that the fusions performed in conjunction with spinal instrumentation were more rigid (P less than .05). Quantitative histomorphometry showed that the volumetric density of bone was significantly lower (ie, device-related osteoporosis occurred) for fused versus unfused spines. In addition, a linear correlation occurred between decreasing volumetric density of bone and increasing rigidity of the spinal implant (r = .778); ie, device-related osteoporosis occurred secondary to Harrington, Cotrel-Dubousset, and Steffee pedicular instrumentation. Oophorectomized dogs became more osteoporotic than their surgically matched controls (posterolateral bone grafting alone, Cotrel-Dubousset pedicular instrumentation, and Steffee pedicular instrumentation); device-related osteoporosis added to the degree of hormonally induced osteoporosis (t = 5.0, P less than .0001). This is the first study to date documenting the occurrence of stress shielding in the spine secondary to spinal instrumentation.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropathologic changes with experimental spinal instrumentation: transpedicular versus sublaminar fixation.

Fifty-six mature beagles underwent lumbar spine destabilization, followed by fusion using four techniques. Spinal cord neuropathologic analysis was carried out to determine the number of abnormalities within each group. Group I (n = 14) had posterolateral bone grafting without instrumentation. Group IIa (n = 14) had Cotrel-Dubousset (CD) pedicle screws and rods. Group IIb (n = 14) had Steffee pedicle screws and plates. Group III (n = 14) had sublaminar wires and rods. All of the animals remained clinically neurologically normal throughout the 6 months of the study. The incidence of moderate to severe neuropathologic changes was 21% in Group I, 18% in Group II, and 64% in Group III. Thus, a significantly higher percentage of neuropathologic abnormalities occurred with sublaminar instrumentation than with no instrumentation (p = 0.027), or with transpedicular instrumentation (p = 0.027). In this controlled animal study, the theoretical advantage of pedicle screws, which should not violate the spinal canal, over sublaminar devices, which must enter the canal, was confirmed.

Biomechanical evaluation of cervical spinal stabilization methods in a human cadaveric model.

The authors have previously reported in vitro testing of various posterior and anterior constructs (sublaminar, Rogers', and Bohlman's triple-wire wiring; AO hook plate fixation; and Caspar anterior plate fixation) in a bovine model with multiaxial biomechanical testing. This study was undertaken to evaluate the above constructs and other constructs in human cadaveric spines. Six subaxial human cervical spine specimens were biomechanically tested at the C5-C6 motion segment both intact and with a simulated distractive-flexion Stage 3 injury created at the C5-C6 level with complete disruption of the supraspinous ligament, interspinous ligament, ligamentum flavum, posterior longitudinal ligament, and facet joint capsules; with sufficient disruption of the intervertebral disc to allow a bilateral C5-C6 facet dislocation. The specimens were tested with a six-channel Bionix MTS 858 materials tester (M.T.S., Minneapolis, Minnesota) using cyclic loads to simulate cervical compression, flexion, extension, and rotation with measurements of axial load, axial displacement, torque, rotation, and anterior and posterior strains. Eight constructs were tested nondestructively: the intact spinal segment, sublaminar wiring, Rogers' wiring, Bohlman's wiring method (triple-wire technique), Roy-Camille posterior plate fixation, AO posterior hook-plate fixation, Caspar anterior plate fixation, and AO posterior hook-plate with Caspar anterior plate fixation. There was no significant difference in flexural stiffness and torsional stiffness between any of the constructs tested; however, there was a significant (P less than 0.05) increase in the posterior strain during flexion and axial loading tests between the Caspar plate construct and all other tested constructs, including the combined posterior and anterior plating construct. These differences persisted after cyclic testing of 100 cycles. Biomechanical testing demonstrated no significant differences between any of the posterior stabilization methods tested. Caspar anterior plating is clearly an inferior method of treating distractive flexion injuries of the cervical spine when compared with all posterior fixation techniques. Also, there is little biomechanical justification for the use of potentially dangerous sublaminar wire fixation and posterior plating methods in these injuries (with intact bony posterior elements), since the relatively safe interspinous wiring methods (Rogers' and Bohlman) are just as rigid as these other posterior fixation techniques.