Surface color spectrophotometry in a murine model of steatosis: an accurate technique with potential applicability in liver procurement.

Steatosis is the most important prognostic histologic feature in the setting of liver procurement. The currently utilized diagnostic methods, including gross evaluation and frozen section examination, have important shortcomings. Novel techniques that offer advantages over the current tools could be of significant practical utility. The aim of this study is to evaluate the accuracy of surface color spectrophotometry in the quantitative assessment of steatosis in a murine model of fatty liver. C57BL/6 mice were divided into a control group receiving normal chow (n = 19), and two steatosis groups receiving high-fat diets for up to 20 weeks-mild steatosis (n = 10) and moderate-to-severe steatosis (n = 19). Mouse liver surfaces were scanned with a hand-held spectrophotometer (CM-600D; Konica-Minolta, Osaka, Japan). Spectral reflectance data and color space values (L*a*b*, XYZ, L*c*h*, RBG, and CMYK) were correlated with histopathologic steatosis evaluation by visual estimate, digital image analysis (DIA), as well as biochemical tissue triglyceride measurement. Spectral reflectance and most color space values were very strongly correlated with histologic assessment of total steatosis, with the best predictor being % reflectance at 700 nm (r = 0.91 [0.88-0.94] for visual assessment, r = 0.92 [0.88-0.95] for DIA of H&E slides, r = 0.92 [0.87-0.95] for DIA of oil-red-O stains, and r = 0.78 [0.63-0.87] for biochemical tissue triglyceride measurement, p < 0.0001 for all). Several spectrophotometric parameters were also independently predictive of large droplet steatosis. In conclusion, hepatic steatosis can accurately be assessed using a portable, commercially available hand-held spectrophotometer device. If similarly accurate in human livers, this technique could be utilized as a point-of-care tool for the quantitation of steatosis, which may be especially valuable in assessing livers during deceased donor organ procurement.

Genetically Defined Oligodendroglioma Is Characterized by Indistinct Tumor Borders at MRI.

BACKGROUND AND PURPOSE: In 2016, the World Health Organization revised the brain tumor classification, making IDH mutation and 1p/19q codeletion the defining features of oligodendroglioma. To determine whether imaging characteristics previously associated with oligodendroglial tumors are still applicable, we evaluated the MR imaging features of genetically defined oligodendrogliomas. MATERIALS AND METHODS: One hundred forty-eight adult patients with untreated World Health Organization grade II and III infiltrating gliomas with histologic oligodendroglial morphology, known 1p/19q status, and at least 1 preoperative MR imaging were retrospectively identified. The association of 1p/19q codeletion with tumor imaging characteristics and ADC values was evaluated. RESULTS: Ninety of 148 (61%) patients had 1p/19q codeleted tumors, corresponding to genetically defined oligodendroglioma, and 58/148 (39%) did not show 1p/19q codeletion, corresponding to astrocytic tumors. Eighty-three of 90 (92%) genetically defined oligodendrogliomas had noncircumscribed borders, compared with 26/58 (45%) non-1p/19q codeleted tumors with at least partial histologic oligodendroglial morphology (P < .0001). Eighty-nine of 90 (99%) oligodendrogliomas were heterogeneous on T1- and/or T2-weighted imaging. In patients with available ADC values, a lower mean ADC value predicted 1p/19q codeletion (P = .0005). CONCLUSIONS: Imaging characteristics of World Health Organization 2016 genetically defined oligodendrogliomas differ from the previously considered characteristics of morphologically defined oligodendrogliomas. We found that genetically defined oligodendrogliomas were commonly poorly circumscribed and were almost always heterogeneous in signal intensity.

Fluorescence in situ hybridization identifies high risk Barrett's patients likely to develop esophageal adenocarcinoma.

Barrett's esophagus (BE) with high-grade dysplasia (HGD) defines a group of individuals at high risk of progression to esophageal adenocarcinoma (EA). Fluorescence in situ hybridization (FISH) has been shown to be useful for the detection of dysplasia and EA in endoscopic brushing specimens from BE patients. The aim of this study was to determine whether FISH in combination with histological findings would further identify more rapid progressors to EA. This is a retrospective cohort study of high-risk patients, having a history of biopsy-confirmed HGD without EA, with an endoscopic brushing specimen analyzed by FISH while undergoing endoscopic surveillance and treatment between April 2003 and October 2010. Brushing specimens were assessed by FISH probes targeting 8q24 (MYC), 9p21 (CDKN2A), 17q12 (ERBB2), and 20q13 (ZNF217) and evaluated for the presence of polysomy, defined as multiple chromosomal gains (displaying ≥ 3 signals for ≥ 2 probes). Specimens containing ≥ 4 cells exhibiting polysomy were considered polysomic. HGD was confirmed by at least two experienced gastrointestinal pathologists. Of 245 patients in this study, 93 (38.0%) had a polysomic FISH result and 152 (62.0%) had a non-polysomic FISH result. Median follow-up was 3.6 years (interquartile range [IQR] 2-5 years). Patients with a polysomic FISH result had a significantly higher risk of developing EA within 2 years (14.2%) compared with patients with a non-polysomic FISH result (1.4%, P < 0.001). These findings suggest that a polysomic FISH result in BE patients with simultaneous HGD identifies patients at a higher risk for developing EA compared with those with non-polysomy.

AQP4 autoantibody assay performance in clinical laboratory service.

OBJECTIVE: To compare performance of contemporary aquaporin-4-immunoglobulin (Ig) G assays in clinical service. METHODS: Sera from neurologic patients (4 groups) and controls were tested initially by service ELISA (recombinant human aquaporin-4, M1 isoform) and then by cell-based fluorescence assays: fixed (CBA, M1-aquaporin-4, observer-scored) and live (fluorescence-activated cell sorting [FACS], M1 and M23 aquaporin-4 isoforms). Group 1: all Mayo Clinic patients tested from January to May 2012; group 2: consecutive aquaporin-4-IgG-positive patients from September 2011 (Mayo and non-Mayo); group 3: suspected ELISA false-negatives from 2011 to 2013 (physician-reported, high likelihood of neuromyelitis optica spectrum disorders [NMOSDs] clinically); group 4: suspected ELISA false-positives (physician-reported, not NMOSD clinically). RESULTS: Group 1 (n = 388): M1-FACS assay performed optimally (areas under the curves: M1 = 0.64; M23 = 0.57 [p = 0.02]). Group 2 (n = 30): NMOSD clinical diagnosis was confirmed by: M23-FACS, 24; M1-FACS, 23; M1-CBA, 20; and M1-ELISA, 18. Six results were suspected false-positive: M23-FACS, 2; M1-ELISA, 2; and M23-FACS, M1-FACS, and M1-CBA, 2. Group 3 (n = 31, suspected M1-ELISA false-negatives): results were positive for 5 sera: M1-FACS, 5; M23-FACS, 3; and M1-CBA, 2. Group 4 (n = 41, suspected M1-ELISA false-positives): all negative except 1 (positive only by M1-CBA). M1/M23-cotransfected cells expressing smaller membrane arrays of aquaporin-4 yielded fewer false- positive FACS results than M23-transfected cells. CONCLUSION: Aquaporin-4-transfected CBAs, particularly M1-FACS, perform optimally in aiding NMOSD serologic diagnosis. High-order arrays of M23-aquaporin-4 may yield false-positive results by binding IgG nonspecifically.

Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-ß-glucuronide of gemfibrozil.

In previous studies, gemfibrozil acyl-ß-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-ß-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-ß-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-ß-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-ß-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.

Limited clinical utility of CT coronary angiography in a district hospital setting.

BACKGROUND: Studies have demonstrated considerable accuracy of multi-slice CT coronary angiography (MSCT-CA) in comparison to invasive coronary angiography (I-CA) for evaluating coronary artery disease (CAD). The extent to which published MSCT-CA accuracy parameters are transferable to routine practice beyond high-volume tertiary centres is unknown. AIM: To determine the accuracy of MSCT-CA for the detection of CAD in a Scottish district general hospital. DESIGN: Prospective study of diagnostic accuracy. METHOD: One hundred patients with suspected CAD recruited from two Glasgow hospitals underwent both MSCT-CA (Philips Brilliance 40 × 0.625 collimation, 50-200 ms temporal resolution) and I-CA. Studies were reported by independent, blinded radiologists and cardiologists and compared using the AHA 15-segment model. RESULTS: Of 100 patients [55 male, 45 female, mean (SD) age 58.0 (10.7) years], 59 and 41% had low-intermediate and high pre-test probabilities of significant CAD, respectively. Mean (SD) heart rate during MSCT-CA was 68.8 (9.0) bpm. Fifty-seven per cent of patients had coronary artery calcification and 35% were obese. Patient prevalence of CAD was 38%. Per-patient sensitivity, specificity, positive and negative (NPV) predictive values for MSCT-CA were 92.1, 47.5, 52.2 and 90.6%, respectively. NPV was reduced to 75.0% in the high pre-test probability group. Specificity was compromised in patients with sub-optimally controlled heart rates, calcified arteries and elevated BMI. CONCLUSION: Forty-Slice MSCT-CA has a high NPV for ruling out significant CAD when performed in a district hospital setting in patients with low-intermediate pre-test probability and minimal arterial calcification. Specificity is compromised by clinically appropriate strategies for dealing with unevaluable studies. Effective heart rate control during MSCT-CA is imperative. National guidelines should be utilized to govern patient selection and direct MSCT-CA reporter training to ensure quality control.

The burden of atrial fibrillation in unselected acute medical admissions.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia and is of increasing prevalence. The presence of AF complicates the management of patients presenting as medical emergencies. OBJECTIVE: To assess the prevalence of AF and current investigation and management strategies in unselected acute medical admissions. DESIGN: Prospective survey of all acute medical admissions over 22 days. SETTING: Stobhill Hospital--district general hospital in north Glasgow. SUBJECTS: Five hundred and seven consecutive acute medical admissions. RESULTS: Of the 507 patients, 47 (9.3%) had AF. AF was a new diagnosis in five patients (11.0%). The most common presenting features were dyspnoea and chest pain. The principal underlying medical conditions were hypertension and ischaemic heart disease. AF was the primary reason for admission in six patients (12.8%) and a documented reason for admission in 11 patients (23.4%). Thyroid function tests were or had previously been performed in 45 patients (95.7%). Twenty-four patients (51.1%) underwent echocardiography or had done so previously. Twenty-two patients (46.8%) received anticoagulation with warfarin. Ten patients (21.3%) should have received warfarin by standard guidelines but did not. No patient received warfarin inappropriately. Rate control was used in 40 patients (85.1%). Rhythm control was attempted in four patients (8.5%). CONCLUSION: AF is common amongst emergency admissions to district general hospitals and has significant resource implications. Improvements are needed both in the use of echocardiography and in the administration of anticoagulant therapy.

Key issues in the management of atrial fibrillation--protecting the patient and controlling the arrhythmia.

Atrial fibrillation (AF) is the most common sustained tachyarrhythmia and its prevalence is increasing. It is an independent risk factor for stroke and is associated with significant morbidity and mortality. AF currently accounts for 1% of NHS expenditure. The management of AF has a broad evidence base and both the American College of Cardiology/American Heart Association/European Society of Cardiology (ACC/AHA/ESC) and the National Institute for Clinical Excellence (NICE) have recently published guidelines. Some controversy persists regarding stroke risk stratification and appropriate anticoagulation regimes although a general consensus is now emerging. Rate and rhythm control strategies have been shown to be comparable in terms of clinical outcomes. Current anti-arrhythmic drugs have limited efficacy and significant side-effect profiles. Electrophysiological and surgical interventions have a role in both strategies. This article broadly reviews the evidence for different management strategies in AF and presents a practical approach to treatment in light of the recently published national and international guidelines.

An initial evaluation of virtual reality simulation in teaching pre-clinical operative dentistry in a UK setting.

The use of virtual reality (VR) training in dentistry is a recent innovation and little research has been conducted to evaluate its use. For each preclinical exercise carried out the VR software currently records a final mark for the procedure, the time taken to complete the procedure and the number of 'internal' assessments carried out by the student. The aims of this study were two fold; a critical appraisal of the software by the students using a structured feedback together with an assessment of any link between the preparation time, final mark and number of evaluations. Sixteen 2nd year undergraduate dental students spent 6 hours cutting an unlimited number of Class I cavities and Class II cavities. The final mark awarded by the VR software together with the overall preparation time and number of evaluations for each cavity were recorded. For the Class I cavity the mean mark obtained was 66.8, the mean preparation time was 12.5 mins and the mean number of evaluations was 6.7. For the Class II cavity the mean mark was 26.5, the mean preparation time was 18 mins and the mean number of evaluations was 7.0. Final marks were also stratified into quartiles (0-24, 25-49, 50- 74, 75-100). For the Class II cavity the time taken to complete the cavity and the number of evaluations made were greater for those cavities that gained a mark of 50 or more. In conclusion, this initial evaluation of the DentSim VR package was a generally positive undergraduate educational experience. The class II cavity was more difficult to cut than the class I which was reflected in the mean scores. There was also a trend towards higher marks being associated with longer preparation times and more evaluations during the preparation.

Dynamic cervical change: is real-time sonographic cervical shortening predictive of preterm delivery in patients with symptoms of preterm labor?

OBJECTIVES: To determine prospectively if dynamic cervical change (spontaneous real-time cervical shortening) is predictive of preterm delivery at < 37 weeks' gestation in patients with symptoms of preterm labor. METHODS: This was a prospective study of patients at 23-34 weeks' gestation who were symptomatic for preterm labor. Patients underwent a 10-min real-time sonographic cervical length assessment with measurements taken at 1-min intervals. The presence or absence of dynamic cervical change, defined as real-time changes in cervical length observable to the naked eye of the sonologist during the examination, was recorded. Gestational age at delivery was obtained from medical records. Preterm delivery was defined as delivery at < 37 weeks' gestation. Dynamic cervical change and initial and minimum cervical lengths were assessed for prediction of preterm delivery. RESULTS: Seventy-six patients were enrolled, and 66 were available for outcome analysis. Thirty-one patients (47%) exhibited dynamic cervical change. Patients with dynamic change had shorter initial cervical lengths (27 mm vs. 36 mm, P = 0.001), shorter minimum cervical lengths (20 vs. 33 mm, P < 0.001) and larger changes in cervical length during the examination period (10 vs. 4 mm, P < 0.001). In the subgroup of patients with an initial cervical length > 30 mm, those with dynamic change delivered earlier than did those without dynamic change (36.8 vs. 38.6 weeks, P = 0.02), and a higher percentage delivered preterm (27% vs. 11%, odds ratio (OR), 3.0 (0.5-17.0)). Multivariate analysis showed that minimum cervical length was a better predictor of preterm delivery than was initial cervical length. CONCLUSIONS: Dynamic cervical change occurs frequently in association with shortened cervical length. In patients with longer initial cervical lengths, dynamic change may increase the risk for preterm delivery. When dynamic change is noted in a patient with preterm labor symptoms, use of the minimum cervical length observed may be better compared with initial cervical length for determining preterm delivery risk.

Dynamic cervical change during real-time ultrasound: prospective characterization and comparison in patients with and without symptoms of preterm labor.

OBJECTIVES: To compare the incidence of dynamic cervical change (spontaneous real-time cervical shortening) in singleton patients with and without symptoms of preterm labor (PTL). METHODS: A total of 109 patients between 23 and 34 weeks' gestation with and without PTL symptoms underwent cervical length ultrasound and contraction monitoring over a 10-min period. Cervical length measurements were taken at 1-min intervals. Exclusion criteria included ruptured membranes, dilation > 3 cm or cerclage. Following the examination, the sonographer made a subjective assessment as to whether noticeable dynamic cervical change had occurred. A measurement was then made during the application of fundal pressure. The initial cervical length, shortest length, maximum change in length and incidence of dynamic change were compared between patients with and without PTL symptoms. The shortest cervical length was compared to the presence and timing of uterine contractions and the measurement during the application of fundal pressure. RESULTS: A total of 43 asymptomatic patients and 66 symptomatic patients were studied. Compared to asymptomatic patients, patients with PTL symptoms had shorter initial lengths, nadir lengths and mean lengths over time as well as a greater amount of maximum change. Dynamic cervical change was more frequently seen in symptomatic patients (48% vs. 9%, P < 0.001) and was associated with uterine contractions (odds ratio 4.6, 95% CI 1.9-10.8). Fundal pressure was not able to reproduce the shortest cervical length that occurred spontaneously during the observation period. CONCLUSIONS: Dynamic cervical change (real-time cervical shortening) is common in patients with PTL symptoms and is associated with uterine contractions. Whether this finding enhances the ability to predict preterm delivery remains to be elucidated.

The impact of biotechnology on agricultural worker safety and health.

Biotechnology applications such as the use and production of genetically modified organisms (GMOs) have been widely promoted, adopted, and employed by agricultural producers throughout the world. Yet, little research exists that examines the implications of agricultural biotechnology on the health and safety of workers involved in agricultural production and processing. Regulatory frameworks do exist to examine key issues related to food safety and environmental protection in GMO applications. However, based on the lack of research and regulatory oversight, it would appear that the potential impact on the safety and health of workers is of limited interest. This article examines some of the known worker health and safety implications related to the use and production of GMOs using the host, agent, and environment framework. The characteristics of employers, workers, inputs, production practices, and socio-economic environments in which future agricultural workers perform various tasks is likely to change based on the research summarized here.

Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments.

The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, betaIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and betaIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. betaIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.

FIGQY phosphorylation defines discrete populations of L1 cell adhesion molecules at sites of cell-cell contact and in migrating neurons.

Phosphorylation of neurofascin, a member of the L1 family of cell adhesion molecules (L1 CAMs), at the conserved FIGQY-tyrosine abolishes the ankyrin-neurofascin interaction. This study provides the first evidence, in Drosophila melanogaster and vertebrates, for the physiological occurrence of FIGQY phosphorylation in L1 family members. FIGQY tyrosine phosphorylation is localized at specialized cell junctions, including paranodes of sciatic nerve, neuromuscular junctions of adult rats and Drosophila embryos, epidermal muscle attachment sites of Drosophila, and adherens junctions of developing epithelial cells of rat and Drosophila. In addition, FIGQY-phosphorylated L1 CAMs are abundantly expressed in regions of neuronal migration and axon extension, including the embryonic cortex, the neonatal cerebellum and the rostral migratory stream, a region of continued neurogenesis and migration throughout adulthood in the rat. Based on our results, physiological FIGQY-tyrosine phosphorylation of the L1 family likely regulates adhesion molecule-ankyrin interactions establishing ankyrin-free and ankyrin-containing microdomains and participates in an ankyrin-independent intracellular signaling pathway at specialized sites of intercellular contact in epithelial and nervous tissue.

Cytoprotection of pancreatic islets before and soon after transplantation by gene transfer of the anti-apoptotic Bcl-2 gene.

Isolated pancreatic islet transplantation is a promising alternative to conventional insulin-dependent diabetes treatment but is not yet a practical clinical therapy. In the first few days after pancreatic islet transplantation, substantial donor pancreatic islet dysfunction and apoptosis commonly occur. Islet apoptosis has been documented after extracellular matrix disruption and exposure to proinflammatory cytokines, and during hypoxia before islet revascularization and rejection. These studies show that targeting the apoptosis pathway by adenoviral-mediated gene transfer of the anti-apoptotic Bcl-2 gene exerts a major cytoprotective effect on isolated macaque pancreatic islets. Bcl-2 transfection ex vivo protects these islets from apoptosis induced by disruption of the islet extracellular matrix during pancreatic digestion. Additionally, overexpression of Bcl-2 confers long-term, stable protection and maintenance of functional islet mass after transplantation of macaque islets into diabetic severe combined immunodeficency mice. Notably, genetic modification of pancreatic islets also reduced the islet mass required to achieve stable euglycemia. Ex vivo gene transfer of anti-apoptotic genes has potential as a therapeutic approach to both minimize loss of functional islet mass after transplant and reduce the high donor islet requirement currently needed for successful stable reversal of insulin-dependent diabetes.

A study of endodontic treatment carried out in dental practice within the UK.

AIM: The purpose of this study was to gather both qualitative and quantitative information on the nature of root canal treatment carried out by a group of dentists working within the United Kingdom. METHODOLOGY: A two-part questionnaire was posted to 720 dentists who graduated from the Dental School, Cardiff, Wales, UK. The first part requested basic information regarding age, year of qualification, field of practice, etc. The second part consisted of 15 questions on endodontic practice and root canal treatment. RESULTS: The response rate was 41.5%. Two hundred and ninety-nine questionnaires contained useful information. The majority of practitioners did not use rubber dam during root canal treatment. The vast majority (89%) exposed a radiograph with an instrument of known length in situ to gauge the 'working length', a small number relied upon tactile sensation. Most practitioners used local anaesthetic solution as an irrigant during instrumentation of the root canal. A wide variety of instruments were used for root canal treatment; a stepback technique was preferred by almost half the practitioners. Antiseptic solution was preferred as an interappointment dressing. More than half of the respondents used laterally condensed gutta-percha to obturate root canals in anterior teeth but only one-third used the same technique in posterior teeth. Less than half the respondents exposed a radiograph to check the fit of the master point prior to obturation. Two-thirds of practitioners used a zinc oxide based material as their root canal sealer. Three-quarters of the practitioners exposed a post obturation radiograph. CONCLUSIONS: The results of this study suggest that although some dentists are using the techniques taught during their undergraduate careers, a large percentage now use techniques with no evidence of clinical effectiveness.

Microtubule/MAP-affinity regulating kinase (MARK) is activated by phenylarsine oxide in situ and phosphorylates tau within its microtubule-binding domain.

Tau is a microtubule-associated protein (MAP) that is functionally modulated by phosphorylation and that is hyperphosphorylated in several neurodegenerative diseases. Because phosphorylation regulates both normal and pathological tau functioning, it is of interest to identify the signaling pathways and enzymes capable of modulating tau phosphorylation in vivo. Previously, it was demonstrated that in SH-SY5Y human neuroblastoma cells and rat primary cortical cultures tau is phosphorylated at Ser262/356, within its microtubule-binding domain, by a staurosporine-sensitive protein kinase in response to the vicinal thiol-directed agent phenylarsine oxide. The current study demonstrates the presence of a 100-kDa protein kinase activity in SH-SY5Y cells that associates with microtubules, phosphorylates tau at Ser262/356, is activated by phenylarsine oxide, and is inhibited by the protein kinase inhibitor staurosporine. Isolation of individual protein bands from a polyacrylamide gel revealed two closely spaced proteins containing Ser262/356-directed protein kinase activity. Mass spectrometry analysis indicated that these protein bands correspond to the 100-kDa microtubule/MAP-affinity regulating kinase (MARK), which has been shown previously to phosphorylate tau within its microtubule-binding domain. Immunoblot analysis of the protein kinase bands confirmed this finding, providing the first demonstration that activation of endogenous MARK results in increased tau phosphorylation within its microtubule-binding domain in situ.

Modulation of tau phosphorylation and intracellular localization by cellular stress.

Tau is a microtubule-associated protein that is functionally modulated by phosphorylation and hyperphosphorylated in several neurodegenerative diseases. Because phosphorylation regulates both normal and pathological tau functioning, it is of great interest to identify the signalling pathways and enzymes capable of modulating tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and localization in response to osmotic stress, which activates the stress-activated protein kinases (SAPKs), a family of proline-directed protein kinases shown to phosphorylate tau in vitro and hypothesized to phosphorylate tau in Alzheimer's disease. Immunoblot analysis with phosphorylation-dependent antibodies revealed that osmotic stress increased tau phosphorylation at the non-Ser/Thr-Pro sites Ser-262/356, within the microtubule-binding domain, as well as Ser/Thr-Pro sites outside of tau's microtubule-binding domain. Although all SAPKs examined were activated by osmotic stress, none of the endogenous SAPKs mediated the increase in tau phosphorylation. However, when transfected into SH-SY5Y cells, SAPK3, but not the other SAPKs examined, phosphorylated tau in situ in response to activation by osmotic stress. Osmotic-stress-induced tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and an increase in the amount of soluble tau. This stress-induced alteration in tau localization was only partially due to phosphorylation at Ser-262/356 by a staurosporine-sensitive, non-proline-directed, protein kinase. Taken together, these results suggest that osmotic stress activates at least two tau-directed protein kinases, one proline-directed and one non-proline-directed, that SAPK3 can phosphorylate tau on Ser/Thr-Pro residues in situ, and that Ser-262/356 phosphorylation only partially regulates tau localization in the cell.

Modulation of tau phosphorylation within its microtubule-binding domain by cellular thiols.

Tau is a microtubule-stabilizing protein that is functionally modulated by alterations in its phosphorylation state. Because phosphorylation regulates both normal and pathological tau functioning, it is of importance to identify the signaling pathways that regulate tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and function in response to modulation of cellular thiol content. Treatment of cells with phenylarsine oxide, which reacts with vicinal thiols, selectively increased tau phosphorylation within its microtubule-binding domain, at the non-Ser/Thr-Pro sites Ser262/356, while decreasing tau phosphorylation at Ser/ Thr-Pro sites outside this region. This increase in tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and decreased microtubule stability. Phenylarsine oxide-induced tau phosphorylation was inhibited by oxidants and by the protein kinase inhibitor staurosporine. Although staurosporine completely eliminated the increase in tau phosphorylation at Ser262/356, as detected by immunostaining with 12E8, it had a comparatively minor effect on the changes in tau localization induced by phenylarsine oxide. The results suggest that regulation of cellular thiols is important for modulating tau phosphorylation and function in situ. Additionally, although phosphorylation of Ser262/356 decreases tau's interaction with the cytoskeleton, phosphorylation of these residues alone is not sufficient for the phenylarsine oxide-induced changes in tau localization.