Shen-Long Howng

Differential expression of Wnt genes, β-catenin and E-cadherin in human brain tumors

Wnt regulates developmental and oncogenic processes through its downstream effector, beta-catenin, and a set of other intracellular regulators that are largely conserved among species. E-cadherin was discovered as a protein associated with beta-catenin which plays a crucial role in cell-cell adhesion. To further understand the molecular basis of Wnt signaling pathway and E-cadherin in brain tumorigenesis, the expression of four Wnt genes (Wnt1, Wnt5a, Wnt10b and Wnt13) and E-cadherin were analyzed by reverse transcriptase-polymerase chain reaction. In addition, their downstream effector, beta-catenin, was also investigated. The results showed that the expression of Wnt5a (41/45), Wnt10b (37/45), and Wnt13 (35/45) were found in brain tumors, whereas Wnt1 (6/45) was shown to be less related. Interestingly, E-cadherin was only expressed in a few cases of astrocytoma (2/16), whereas it was expressed in most meningioma (14/15) and pituitary adenoma tumors (12/14). There was no apparent difference of beta-catenin expression profile in brain tumors; however, the sequencing data of beta-catenin showed two mutations on speculative phosphorylation sites, S73F and S23G in astrocytoma. Furthermore, an in vitro functional assay showed that S73F and S23G mutants of beta-catenin did not affect transcriptional activity in TCF-4-leuciferase reporter construct, suggesting that they may need more complex factors to participate in astrocytoma. Taken together, our data suggest that the mutations of beta-catenin together with E-cadherin and Wnt signaling might be involved in brain tumorigenesis.

The conditional probabilities of survival in patients with anaplastic astrocytoma or glioblastoma multiforme

BACKGROUND By the use of conditional probabilities of survival, we studied the yearly survival rates for individual tumor survivors. METHODS Conditional survival rate was estimated in 114 consecutive patients with anaplastic astrocytoma or glioblastoma multiforme. Conditional probabilities of surviving some years given survival to a specific period of time after craniotomy and 95% confidence intervals were calculated in the individual tumor survivors. RESULTS The estimated median survival was 30 months for 45 patients with anaplastic astrocytoma and 12 months for 69 patients with glioblastoma multiforme. The conditional probabilities of surviving next one year given survival to 1 year, 2 years, 3 years, 4 years, or 5 years after craniotomy for anaplastic astrocytoma were 86.2%, 75.0%, 85.9%, 77.8%, or 85.7%, respectively; for glioblastoma multiforme 64.8%, 58.7%, 85.7%, 80.0%, or 75.0%, respectively. The conditional probability of surviving to 5 years given survival to 2 years after craniotomy for anaplastic astrocytoma, i.e., surviving an additional 3 years, was 50.1%, which was better than observed 5-year survival rate (28.6%); for glioblastoma multiforme it was 40.2%, which also was better than observed 5-year survival rate (12.4%). CONCLUSIONS The conditional probability of survival was a good method to clinically predict yearly survival rate for individual tumor survivors. In addition, the method can estimate the probabilities of surviving next some years given survival to a specific period of time after craniotomy. It also showed a more encouraging result than observed survival rate in patients with supratentorial malignant astrocytomas.

GSK3beta-Mediated Drp1 Phosphorylation Induced Elongated Mitochondrial Morphology against Oxidative Stress

Multiple phosphorylation sites of Drp1 have been characterized for their functional importance. However, the functional consequence of GSK3beta-mediated phosphorylation of Drp1 remains unclear. In this report, we pinpointed 11 Serine/Threonine sites spanning from residue 634∼736 of the GED domain and robustly confirmed Drp1 Ser693 as a novel GSK3beta phosphorylation site. Our results suggest that GSK3beta-mediated phosphorylation at Ser693 does cause a dramatic decrease of GTPase activity; in contrast, GSK3beta-mediated phosphorylation at Ser693 appears not to affect Drp1 inter-/intra-molecular interactions. After identifying Ser693 as a GSK3beta phosphorylation site, we also determined that K679 is crucial for GSK3beta-binding, which strongly suggests that Drp1 is a novel substrate for GSK3beta. Thereafter, we found that overexpressed S693D, but not S693A mutant, caused an elongated mitochondrial morphology which is similar to that of K38A, S637D and K679A mutants. Interestedly, using H89 and LiCl to inhibit PKA and GSK3beta signaling, respectively, it appears that a portion of the elongated mitochondria switched to a fragmented phenotype. In investigating the biofunctionality of phosphorylation sites within the GED domain, cells overexpressing Drp1 S693D and S637D, but not S693A, showed an acquired resistance to H2O2-induced mitochondrial fragmentation and ensuing apoptosis, which affected cytochrome c, capase-3, -7, and PARP, but not LC3B, Atg-5, Beclin-1 and Bcl2 expressions. These results also showed that the S693D group is more effective in protecting both non-neuronal and neuronal cells from apoptotic death than the S637D group. Altogether, our data suggest that GSK3beta-mediated phosphorylation at Ser693 of Drp1 may be associated with mitochondrial elongation via down-regulating apoptosis, but not autophagy upon H2O2 insult.

Acute subdural hematoma in infancy.

BACKGROUND Acute subdural hematoma in infants is distinct from that occurring in older children or adults because of differences in mechanism, injury thresholds, and the frequency with which the question of nonaccidental injury is encountered. The purpose of this study is to analyze the clinical characteristics of acute subdural hematoma in infancy, to discover the common patterns of this trauma, and to outline the management principles within this group. METHODS Medical records and films of 21 cases of infantile acute subdural hematoma were reviewed retrospectively. Diagnosis was made by computed tomography or magnetic resonance imaging. Medical records were reviewed for comparison of age, gender, cause of injury, clinical presentation, surgical management, and outcome. RESULTS Twenty-one infants (9 girls and 12 boys) were identified with acute subdural hematoma, with ages ranging from 6 days to 12 months. The most common cause of injury was shaken baby syndrome. The most common clinical presentations were seizure, retinal hemorrhage, and consciousness disturbance. Eight patients with large subdural hematomas underwent craniotomy and evacuation of the blood clot. None of these patients developed chronic subdural hematoma. Thirteen patients with smaller subdural hematomas were treated conservatively. Among these patients, 11 developed chronic subdural hematomas 15 to 80 days (mean = 28 days) after the acute subdural hematomas. All patients with chronic subdural hematomas underwent burr hole and external drainage of the subdural hematoma. At follow-up, 13 (62%) had good recovery, 4 (19%) had moderate disability, 3 (14%) had severe disability, and 1 (5%) died. Based on GCS on admission, one (5%) had mild (GCS 13-15), 12 (57%) had moderate (GCS 9-12), and 8 (38%) had severe (GCS 8 or under) head injury. Good recovery was found in 100% (1/1), 75% (8/12), and 50% (4/8) of the patients with mild, moderate, and severe head injury, respectively. Sixty-three percent (5/8) of those patients undergoing operation for acute subdural hematomas and 62% (8/13) of those patients treated conservatively had good outcomes. CONCLUSIONS Infantile acute subdural hematoma if treated conservatively or neglected, is an important cause of infantile chronic subdural hematoma. Early recognition and suitable treatment may improve the outcome of this injury. If treatment is delayed or the condition is undiagnosed, acute subdural hematoma may cause severe morbidity or even fatality.

Cloning and characterization of a novel human ninein protein that interacts with the glycogen synthase kinase 3β

Using human glycogen synthase kinase 3beta (GSK-3beta) as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, hNinein. When the full length cDNA of hNinein was sequenced, it showed that an open reading frame encoded a protein consisting of 2047 amino acids with a predicted molecular mass of 239 kDa. The features of this protein include a potential GTP binding site, a large coiled-coil domain together with four leucine zipper domains and a GSK-3beta binding site. Fluorescence microscopy experiment showed that hNinein is localized in the pericentriolar matrix of the centrosome. In addition, hNinein also showed to react with centrosomal autoantibody sera. Our findings suggest that hNinein may be involved in the formation of centrosome matrix and interacts with the GSK-3beta, implying that it may also be regulated by GSK-3beta phosphorylation signaling.

Outcome analyses of interbody titanium cage fusion used in the anterior discectomy for cervical degenerative disc disease.

Anterior discectomy and fusion to treat cervical degenerative disc disease is the preferred procedure for many spine surgeons. The ideal device for structural reconstruction of the anterior cervical spine remains controversial. The purpose of this prospective study was to investigate the effectiveness of a non-threaded titanium cage in performing anterior spinal fusion for cervical degenerative disc disease. The clinical and radiologic data of 78 consecutive patients were reviewed. Neurologic outcome was assessed using Odoms criteria. Neck pain was graded using a 10-point visual analog scale. The cervical spinal curvature, the height of foramina, and fusion status were evaluated on preoperative and postoperative radiographs. Mean follow-up was 24.9 (range 18-35) months. An excellent or good result was found in 92% of the patients with radiculopathy, 69% of those with myelopathy, and 73% of those with myeloradiculopathy. Statistical analyses also showed improvement of cervical pain after surgery (P < 0.001) and a significant increase in foraminal height (P = 0.035). Cervical kyphosis was present in 27 (34%) patients before surgery; it was corrected to lordosis in 9. The fusion rate at 12 months and 24 months was 91% and 95%, respectively. No surgery or cage-related complication occurred in these patients. Non-threaded interbody cage fusion in this study achieved a high fusion rate and had a good neurologic outcome. These results suggest that non-threaded cage fusion is a safe and effective method for anterior cervical discectomy.

Simultaneous multiple hypertensive intracerebral haemorrhages

Background.Simultaneous occurrence of multiple intracerebral haemorrhages (ICHs) in different arterial territories is a rare clinical event which has been reported to be associated with cerebral amyloid angiopathy, venous sinus thrombosis, coagulopathy, vasculitis, haemorrhagic transformation of cerebral infarcts and multiple intracranial pathologies such as vascular anomalies or tumours. Although hypertension is the most common etiological factor for the development of spontaneous single intracerebral bleeding, its role in simultaneous multiple ICHs is not clear.Methods. The authors have reviewed all patients with non-traumatic ICH admitted to Kaohsiung Medical University Hospital from 1993 to 2002. Ten hypertensive patients with simultaneous multiple ICHs were found. For the purpose of comparison, another 600 cases with solitary hypertensive ICH were also reviewed as a control group. Computerized tomographic scans and medical records concerning patients’ histories, clinical presentations, locations of haematomas, associated risk factors, and outcome were analyzed.Findings. The mean age and sex distribution were similar in both patient groups. Bilateral putaminal or thalamic haemorrhages were the most common combinations of simultaneous bleedings. As for the individual location of haematoma, there was a strong preponderance for the supratentorial space with the thalamus being the most preferable site. The duration of hypertension was longer and the percentage of previous stroke was higher in patients with multiple ICHs. Other associated risk factors were similar in both groups except for higher incidence of hypercholesterolemia in multiple ICHs group. Patients with simultaneous multiple ICHs had a much worse outcome compared to those with solitary ICH.Conclusions. As with solitary ICH, hypertension is still the most important etiological factor for simultaneous multiple ICHs. The widespread and prolonged degeneration of intracerebral arterioles predispose patients to the development of multiple ICHs, which could be justified by the longer history of hypertension and higher incidence of former strokes. Only hypercholesterolemia was identified to be significantly associated with this unusual brain event in our study. The mechanism underlying the development of simultaneous multiple ICHs is not clear although structural and haemodynamic changes of first haemorrhage may be responsible for the second one. Poorer outcome in patients with multiple ICHs can be explained by the concomitant destruction of crossing and non-crossing fiber tracts and bilateral diaschisis phenomenon.

Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle☆

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303-2096) co-localized with gamma-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783-1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.

Factors influencing seizures in adult patients with supratentorial astrocytic tumors

SummarySeizures and epilepsy in adults are important and increasingly common clinical problems. Despite this, the investigation of seizures in adults with astrocytic tumors remains a grey area. The incidence and influencing factors of preoperative and postoperative seizures were evaluated in 101 patients of 45 years or older with supratentorial astrocytic tumors. Preoperative seizures occurred in 14 (14%) patients. Seizures at presentation were significantly correlated with pathological grades of astrocytic tumors (p=0.0318). The risk of seizures at presentation was greatest in patients with well-differentiated astrocytomas as compared with anaplastic astrocytomas (Odds ratio=4.364, p=0.056) or glioblastomas multiforme (Odds ratio=5.673, p=0.007). There was no association of preoperative seizures with age, sex, location or site of the tumors. Postoperative seizures occurred in 18 (18%) patients, including 8 (8/14, 57%) recurrent seizures and 10 (10/87, 12%) late-onset seizures. Postoperative seizures were significantly correlated with the presence of preoperative seizures (p=0.0003). The presence of preoperative seizures was potentially predictive of postoperative seizures when evaluated by logistic regression model (Odds ratio=6.650). Thirteen (72%) of 18 patients with postoperative seizures were associated with tumor recurrence in 7 cases, hemorrhage in 3 cases and malignant progression in 3 cases. There was no association of postoperative seizures with age, sex, location or site of the tumors, grades of tumors, type of preoperative seizures, duration of preoperative seizures, serum level of anticonvulsant drug, extent of surgery, postoperative radiation or chemotherapy. The patients with preoperative seizures had a higher risk of postoperative seizures and should be carefully monitored. Imaging examination of brain to exclude the possibilities of tumor recurrence or hemorrhage is warrantable in supratentorial astrocytoma patients with postoperative seizures.

A novel ninein‐interaction protein, CGI‐99, blocks ninein phosphorylation by GSK3β and is highly expressed in brain tumors

To explore more hNinein interacting proteins, the yeast two‐hybrid screening using ninein C‐terminal domain as bait protein was performed. One novel gene, CGI‐99, was demonstrated to associate with hNinein in the yeast two‐hybrid method and in vitro GST pull‐down assay. Molecular characterization also showed that CGI‐99 possessed a transcriptional activity at the N‐terminal. In addition, CGI‐99 formed a dimer with the C‐terminal, which overlapped with hNinein binding site. In kinase assay, CGI‐99 binds to hNinein and completely blocks the phosphorylation of hNinein by GSK3β. Moreover, CGI‐99 was highly expressed in all brain tumors which is in agreement with the Northern blot analysis. Taken together, we have isolated a novel protein CGI‐99, which may be involved in the functional regulation of human ninein in the centrosome structure and may also be important in brain development and tumorigenesis.