Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD.

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)-induced cell death. We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.

A decade of site- and phosphorylation state-specific antibodies: recent advances in studies of spatiotemporal protein phosphorylation.

From 1990 to 2001, numerous site- and phosphorylation state-specific antibodies have been developed and many are now commercially available. These antibodies have facilitated understanding of the cytoskeletal organization, signal transduction and transcriptional mechanisms as well as clinical diseases. This review is an attempt to cover all these aspects.

Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein.

To elucidate the function of keratins 8 and 18 (K8/18), major components of the intermediate filaments of simple epithelia, we searched for K8/18-binding proteins by screening a yeast two-hybrid library. We report here that human Mrj, a DnaJ/Hsp40 family protein, directly binds to K18. Among the interactions between DnaJ/Hsp40 family proteins and various intermediate filament proteins that we tested using two-hybrid methods, Mrj specifically interacted with K18. Immunostaining with anti-Mrj antibody showed that Mrj colocalized with K8/18 filaments in HeLa cells. Mrj was immunoprecipitated not only with K18, but also with the stress-induced and constitutively expressed heat shock protein Hsp/c70. Mrj bound to K18 through its C terminus and interacted with Hsp/c70 via its N terminus, which contains the J domain. Microinjection of anti-Mrj antibody resulted in the disorganization of K8/18 filaments, without effects on the organization of actin filaments and microtubules. Taken together, these results suggest that Mrj may play an important role in the regulation of K8/18 filament organization as a K18-specific co-chaperone working together with Hsp/c70.

p21-activated kinase PAK phosphorylates desmin at sites different from those for Rho-associated kinase.

p21-activated kinase (PAK) and Rho-associated kinase (Rho-kinase) have been shown to induce Ca(2+)-independent contraction of smooth muscle. PAK-induced contraction of Triton-skinned smooth muscle correlates with increased phosphorylation of caldesmon and desmin, although the role of desmin phosphorylation has remained obscure. Here we report that desmin serves as an excellent substrate for PAK in vitro. PAK phosphorylated desmin in a GTP. Cdc42/Rac-dependent manner. Phosphorylation of desmin by PAK dramatically inhibited its filament-forming ability. PAK phosphorylated mainly serine residues of the head domain of desmin, and the major phosphorylation sites differed from those for Rho-kinase. These results suggest that different site-specific phosphorylation of desmin via two divergent protein kinases downstream of Rho family GTPases would seem to increase the regulatory potential for organization of desmin filaments.

Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin- mediated cell-cell adhesion.

The small guanosine triphosphatases (GTPases) Cdc42 and Rac1 regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a target of Cdc42 and Rac1, was localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L fibroblasts expressing E-cadherin (EL cells), and interacted with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induced the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells, but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, resulted in a decrease in E-cadherin-mediated cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion through the cadherin-catenin pathway.

Overexpression of p21WAF1/CIP1 induces cell differentiation and growth inhibition in a human glioma cell line.

p21WAF1/CIP1 is a downstream mediator of p53 and mediates growth arrest by inhibiting the action of G1 cyclin-dependent kinases. Since cellular differentiation is frequently characterized by G1 arrest, we examined whether p21WAF1/CIP1 overexpression would induce growth suppression and differentiation in p53-defective human glioma cells. Overexpression of p21WAF1/CIP1 resulted in an accumulation of cells in G1, altered morphology, growth arrest and cell differentiation. The extent of cell differentiation correlated with the level of p21WAF1/CIP1 as well as of proliferating cell nuclear antigen, cyclin E, and cdk 2, which associates with p21WAF1/CIP1. Our data suggest that gene transfer of p21WAF1/CIP1 may arrest glioma cell growth in vivo by committing malignant glioma cells to a pathway of terminal differentiation.

Possible involvement of the inactivation of the Rho-Rho-kinase pathway in oncogenic Ras-induced transformation.

Recent evidence has strongly suggested the involvement of Rho family small guanosine triphosphatases (GTPases) in Ras-induced transformation. To further clarify the role of Rho family GTPases in Ras-induced transformation, we examined the effects of dominant active or dominant negative forms of Rho family GTPases on the morphological changes induced by oncogenic Ras (RasV12) in Rat1 fibroblasts. The cells expressing RasV12 showed the severe disruption of actin stress fibers and cell adhesions. The coexpression of dominant active form of Rho (RhoV14) reverted not only the formation of stress fibers and focal adhesions but also cell-cell adhesions in Ras-transformed Rat1 cells. In addition, the coexpression of constitutively activated Rho-kinase, a downstream effector of Rho, restored the assembly of stress fibers and focal adhesions. Treatment of Ratl cells with lysophosphatidic acid, which is known to activate the Rho-Rho-kinase pathway, enhanced the stress fiber formation, whereas it failed to induce the stress fiber formation in the cells expressing RasV12. These results suggest that the Rho-Rho-kinase pathway may be inactivated in the cells expressing RasV12, and this may contribute to oncogenic Ras-induced transformation.

Regulation of cell-cell adhesion of MDCK cells by Cdc42 and Rac1 small GTPases.

Rac1, a member of the Rho small GTPases family, has recently been shown to be involved in the regulation of cell-cell adhesion mediated by cadherin. Here we showed that Cdc42, another member of Rho family, accumulated at cell-cell contact sites. Microinjection of Rho GDI, a negative regulator of the Rho family members, into Madin-Darby canine kidney (MDCK) cells resulted in perturbation of epithelial cell morphology and of cell-cell and cell-substratum adhesions, and comicroinjection of dominant active Cdc42 or Rac1 reversed the action of Rho GDI, suggesting that the active form of Cdc42 or Rac1 is required for maintaining the cell-cell and cell-substratum adhesions. These observations suggest that Cdc42, in addition to Rac1, can regulate the cell-cell adhesion.

Phosphorylation of neurofibromatosis type 1 gene product (neurofibromin) by cAMP-dependent protein kinase.

The critical function of the neurofibromatosis type 1 (NF1) gene product (neurofibromin) is not well defined except that neurofibromin has homology with a family of the GTPase-activating proteins (GAPs). In this study, we confirmed that neurofibromin is constitutively phosphorylated and detected kinase activities which specifically phosphorylate the cysteine/serine-rich domain and the C-terminal domain of the neurofibromin in cell lysate. In vitro and in-gel kinase assays strongly indicated that cAMP-dependent protein kinase (PKA) is a candidate for the neurofibromin kinase. THe biological significance of the phosphorylation of neurofibromin is unclear at present, but we speculate that neurofibromin plays a crucial role in cellular function since it links the two major cellular pathways which are the GAP-ras and PKA-associated signals.

Detection of cellular proteins that interact with the NF2 tumor suppressor gene product.

The neurofibromatosis type 2 (NF2) gene was recently cloned, and the protein it encodes (merlin) was revealed to belong to a family of proteins that link cytoskeletal components with proteins in the cell membrane. To elucidate the biological function of merlin, we produced a bacterial fusion protein consisting of glutathione S-transferase and merlin and used it to detect five merlin-binding cellular proteins, designated p165, p145, p125, p85 and p70, by a protein-binding assay. p165 and merlin were phosphorylated on serine/threonine residues, and immunoprecipitation showed that p85 bound the native form of merlin. Although the entire merlin-ezrin-radixin-moesin (MERM) homology domain of merlin was found to be essential for binding to all five proteins, the MERM homology domains of ezrin and moesin did not bind to any of the five proteins. Since most reported NF2 mutations are in the region we determined was necessary for binding, the mutations probably impair binding. Therefore, the formation of the protein complex is probably crucial for tumor suppression.

Congenital frontal bone defect with intact overlying scalp.

An unusual case of a congenital frontal bone defect with intact overlying scalp and intact underlying dura mater is reported. Although spontaneous healing by the intact underlying dura mater by regeneration was expected, it did not occur. Cranioplasty was done for protective and cosmetic purposes. A review of the literature of congenital skull defects shows that spontaneous regeneration does not occur in this rare anomaly.

Intraorbital arteriovenous malformation: case report.

A case of intraorbital arteriovenous malformation presenting with visual loss, exophthalmos, and chemosis of the right eye is reported. Enhanced computer tomography and magnetic resonance imaging showed extraocular muscle enlargement and vascular abnormality in the right retrobulbar space. Angiography revealed an abnormal intraorbital vascular stain with an extremely dilated right ophthalmic artery. Total removal of the intraorbital contents was performed after unsuccessful endovascular and surgical treatment of arteriovenous malformation (AVM). Histopathological examination disclosed an AVM in the retrobulbar fatty tissue with extension into the extraocular muscles and optic nerve.

Bacterial expression of an active tyrosine kinase from a protein A/truncated c-src fusion protein.

The carboxy-terminal half of the c-src protein fused to the protein A moiety was expressed in bacteria. The protein A/truncated c-src fusion protein, which does not have SH2 and SH3 domains, is found in the periplasmic space allowing for a simple one-step purification and demonstrated high efficiency in autophosphorylation and exogeneous substrate phosphorylation. The missense mutation at codon 294 (Ile-->Thr), which is located in the ATP-binding domain of the c-src, resulted in dramatic reduction of tyrosine kinase activity of the fusion protein. Using the fusion protein, we also revealed that staurosporin, a well-known kinase inhibitor, directly affects autophosphorylation of the C-terminal half of the c-src protein. This truncated c-src expression system provides a good source of enzyme for diverse experiments and is an ideal model for understanding the implication of structural alterations in the catalytic activity of the c-src kinase by site-directed mutagenesis experiments.

Treatment of early recurrent medulloblastoma in children with cisplatin and etoposide: a preliminary report.

The prognosis of recurrent medulloblastoma remains extremely poor. Combination chemotherapy with cisplatin (CDDP) and etoposide (VP-16) was given to five children with early recurrent medulloblastoma. As a rule, CDDP 20 mg/m2 per day and VP-16 60 mg/m2 per day were administered intravenously for 5 days. This cycle was repeated three times at 4-week intervals. After this therapy, cerebellar signs improved in one case and were unchanged in four cases. Weakness and sensory disturbance, however, improved in three of four patients. Moreover, neck and/or back pain resolved in all these four. Radiological findings improved in three cases. Myelosuppression appeared in all patients, but receded rapidly. No other significant complications were noticed. Two patients died 5 and 6 months after this therapy. These results seem to suggest that this therapy has a use in improving neurological symptoms, particularly neck and/or back pain, although its efficacy is limited.

Chronic subdural hematoma in elderly people: present status on Awaji Island and epidemiological prospect.

The epidemiological aspect of chronic subdural hematoma (CSH) in the elderly who are 65 years old or elder was evaluated on Awaji Island with about 170,000 inhabitants. The overall incidence of CSH was 13.1 per 100,000/year, 3.4 in people under 65 years old, and 58.1 in the elderly. The elderly were 17.7% of all inhabitants. If these incidences of CSH are extrapolated to all of Japan in the year 2020, the incidence will be 16.3 per 100,000/year. This suggests that CSH may become the most common neurosurgical condition.

Multiple cerebral and pulmonary arteriovenous malformations in association with brain and subcutaneous abscesses: a possible variant of hereditary hemorrhagic telangiectasia--case report.

The authors present a case in which multiple cerebral and pulmonary arteriovenous malformations (AVMs), a brain abscess, and a recurrent subcutaneous abscess were found concurrently in a 52-year-old male. He was admitted to our hospital for evaluation of a subcutaneous abscess in the right nuchal area and a right occipital AVM that had been detected at another hospital. Of his eight siblings, three had died of cerebrovascular disease (one in childhood and two as young adults), one had died of unknown causes in childhood, one had been hemiplegic since infancy, one had recently undergone removal of a pulmonary AVM, one was killed in World War II at the age of 24 years, and the remaining sibling was healthy. He had had surgery for a right occipital brain abscess three years prior to this admission. A general examination revealed no abnormalities other than the painful right nuchal mass. Neurological evaluation disclosed left homonymous hemianopsia, which was probably a result of his previous surgery for the right occipital brain abscess. Cerebral angiography showed AVMs in the right parietal (2 x 2 cm), right occipital (1.5 x 1.5 cm), and right cerebellar areas (1 x 1 cm). Digital subtraction angiography of the lung revealed multiple bilateral AVMs. The cerebral and pulmonary AVMs were removed in a two-stage operation. Although this case did not correspond precisely to the triad of hereditary hemorrhagic telangiectasia (HHT), the authors consider it to be related to HHT, since that syndrome is often complicated by multiple cerebral and pulmonary AVMs.

[Intramedullary invasion by a cervical cystic neurinoma--a case report].

This report examines a case involving a huge cystic cervical neurinoma existing extra- and intramedullary. The histogenesis of intramedullary neurinoma, pathohistogenesis of large intratumoral cyst are discussed. The role of Magnetic Resonance Imaging (MRI) in the diagnosis of spinal tumors is also discussed. A 23-year-old male was admitted to our ward or having tetraparesis. On admission, spastic tetraparesis and sensory disturbance below level C 2 were noticed. Electrophysiological examinations suggested a left-dominant intraspinal lesion. Conventional radiological examinations revealed widened cervical spinal canal, swelling of the spinal cord at level C 6-7, left extramedullary mass at level C 5, and a complete block at level C 4. MRI disclosed intramedullary tumor existing from the medulla oblongata to the lower cervical including macrocysts, and an extramedullary tumor on the left at level C 3-5. Surgical exploration was made and both of intra- and extramedullary tumors were confirmed to be neurinoma. The postoperative course was favourable. The patient was able to walk with aids, and was referred to the rehabilitation center 6 months after the operation. In histological investigations, the major components of the tumors were typically Antoni-A type neurinoma, and an abundant hemosiderin deposits. There were many hyalinized whorling portions around the cysts. Though spinal neurinoma is the most common spinal tumor, the intramedullary neurinoma is extremely rare. Only 31 cases have been reported as far as we could investigated. The histogenesis of this type of lesion is still unsettled.(ABSTRACT TRUNCATED AT 250 WORDS)

[Hydrocephalus and vasospasm after subarachnoid hemorrhage from ruptured intracranial aneurysms].

The relationship of the amount of subarachnoid blood to the incidence of acute hydrocephalus, delayed vasospasm, and chronic hydrocephalus was investigated in 47 patients with subarachnoid hemorrhage from ruptured intracranial aneurysms. Acute hydrocephalus, delayed vasospasm, and chronic hydrocephalus occurred in 29%, 7%, and 14% of Fisher Group 1 & 2 patients respectively, in contrast, 70%, 64%, and 58% of Fisher Group 3 patients (p less than 0.01). Thirty six percents of all patients had both acute hydrocephalus and vasospasm; Thirty two percents had neither. Twenty one percents had acute hydrocephalus, but no spasm; Eleven percents had spasm, but no acute hydrocephalus. Acute hydrocephalus and vasospasm were significantly associated (p less than 0.05). Most of patients with chronic hydrocephalus showed severe subarachnoid hemorrhage on initial CT scan, followed acute hydrocephalus, and highly associated with vasospasm. These sequelae of SAH are closely linked, mainly by the presence of subarachnoid clot, but there may be some direct causal relationship between them.

[Clinical effects of lipo-prostaglandin E1 in patients with delayed cerebral vasospasm].

Prostaglandin (PG) E1 is a potent vasodilator on the peripheral vessels and also has an inhibitory action of platelet aggregation. Thus it is expected that PGE1 may be used for the treatment of cerebral vasospasm in aneurysmal subarachnoid hemorrhage. Lipo-PGE1, the lipid emulsified PGE1, is not destroyed in the lung, has much longer half life in the circulation than PGE1 which is rapidly inactivated in the lung. The effects of intravenous injection of lipo-PGE1 on the cerebral hemodynamics and the central conduction time (CCT) of the sensory evoked potential under vasospastic conditions has been studied in eight patients. All these 8 patients demonstrated severe angiographic vasospasm and signs of cerebral ischemia. The 15-20 micrograms of lipo-PGE1 was administered every eight hours for 5 to 7 days. Within 6 hours of the first lipo-PGE1 treatment, the regional cerebral blood flow (rCBF) and the CCT measurements were reported to document the effect of treatment. The average pretreatment of rCBF on the right anterior, middle and posterior cerebral artery were 46.6 +/- 6.8, 56.4 +/- 7.3 and 58.8 +/- 8.9 ml/100 g/min and on the left side were 52.1 +/- 9.2, 49.1 +/- 10.8 and 56.1 +/- 9.2 ml/100 g/min respectively. With treatment these flows increased to 53.0 +/- 6.9, 64.3 +/- 5.3 and 63.0 +/- 4.6 ml/100 g/min respectively on the right side and 60.8 +/- 9.4, 60.6 +/- 9.7 and 60.6 +/- 7.2 ml/100 g/min respectively on the left. The CCT also demonstrated the improvement from 6.36 msec to 6.21 msec by the initial PGE1 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)