Endoscopic hematoma evacuation for acute subdural hematoma in a young patient: a case report.

Case: The standard treatment for acute subdural hematoma (ASDH) is large craniotomy; decompressive craniectomy may also be carried out, if needed, to prevent secondary brain damage. Recently, an endoscopic procedure for elderly patients with ASDH was carried out and reported; its safety and effectiveness were emphasized because of minimal invasiveness. We report a young case and discuss its difficulties and tips.A 31-year-old man was found to be in a state of general convulsion. At the time of admission, we observed severe consciousness disturbance, anisocoria, and left hemiparesis. Computed tomography showed a massive subdural hematoma with marked midline shift. Outcome: Osmotherapy and emergency trepanation improved anisocoria. An endoscopic procedure under local anesthesia was sequentially selected. After surgery, the patient's symptoms clearly improved. Conclusion: Although the standard treatment for ASDH is craniotomy, endoscopic surgery may be useful in some cases.

Direct oxidative conversion of methylarenes into aromatic nitriles.

A variety of methylarenes were successfully converted into the corresponding aromatic nitriles in good to moderate yields by the treatment with NBS or DBDMH in the presence of a catalytic amount of AIBN or BPO, followed by the reaction with molecular iodine in aq NH3 in a one-pot procedure. The present reaction is a useful and practical transition-metal-free method for the preparation of aromatic nitriles from methylarenes.

[Observation of de novo formation and growth of an intracranial aneurysm: a case report].

We observed a de novo formation and growth of an aneurysm in a 43-year-old woman who was followed up after treatment of a subarachnoid hemorrhage (SAH). In 2002, the patient, whose mother had a history of SAH, presented with SAH at the age of 36. Three-dimensional computed tomography angiography (3D-CTA) and digital subtraction angiography showed an aneurysm in the right internal carotid-posterior communicating artery. The aneurysm was clipped and postoperative course was uneventful without neurological deficit. The patient was followed up by 3D-CTA and magnetic resonance angiography every 6 months, because of an untreated small aneurysm, 3 mm in diameter, in the left middle cerebral artery (MCA). The MCA aneurysm remained unchanged but a de novo aneurysm, 1.5 mm in diameter, developed in the right anterior cerebral artery (ACA) 6 years after the first surgery. The ACA aneurysm grew to 4 mm in diameter during the following 10 months but the MCA aneurysm remained unchanged. Both aneurysms were clipped in one session. The MCA aneurysm had a smooth wall but the ACA aneurysm had an irregular and thin wall. The postoperative course was uneventful. Young female patients who have developed SAH with familial history, like this case, should receive long-term follow up to check whether a de novo aneurysm has developed.

Characterization of RNase HII substrate recognition using RNase HII-argonaute chimaeric enzymes from Pyrococcus furiosus.

RNase H (ribonuclease H) is an endonuclease that cleaves the RNA strand of RNA-DNA duplexes. It has been reported that the three-dimensional structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) protein, although the two enzymes share almost no similarity in their amino acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition. In contrast, prokaryotic Ago proteins show greater affinity for RNA-DNA hybrids than for RNA-RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, RNase HII) digests RNA-RNA duplexes in the presence of Mn2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf-Ago, based on their protein secondary structures. We found that one of the conserved secondary structural regions (the fourth beta-sheet and the fifth alpha-helix of Pf-RNase HII) contains family-specific amino acid residues. Using a series of Pf-RNase HII-Pf-Ago chimaeric mutants of the region, we discovered that residues Asp110, Arg113 and Phe114 are responsible for the dsRNA (double-stranded RNA) digestion activity of Pf-RNase HII. On the basis of the reported three-dimensional structure of Ph-RNase HII from Pyrococcus horikoshii, we built a three-dimensional structural model of RNase HII complexed with its substrate, which suggests that these amino acids are located in the region that discriminates DNA from RNA in the non-substrate strand of the duplexes.

Site-specific unglycosylation to improve crystallization of the metabotropic glutamate receptor 3 extracellular domain.

Metabotropic glutamate receptors (mGluRs) are involved in the regulation of many physiological and pathological processes in the central nervous system. The extracellular domain (ECD) of mGluR subtype 3 (mGluR3) was produced using the baculovirus expression system and purified from the culture medium. However, the recombinant protein showed heterogeneity in molecular weight on SDS-PAGE analysis. It was found that the unglycosylation of Asn414 significantly reduced the heterogeneity. Consequently, three site-specifically unglycosylated mutant proteins of mGluR3 ECD, replacing Asn414 only or replacing Asn414 in combination with other glycosylation sites, were successfully crystallized in the presence of L-glutamate. Among them, crystals of the N414/439Q mutant diffracted X-rays to 2.35 A resolution using synchrotron radiation. The crystal belonged to the monoclinic space group P2(1), with unit-cell parameters a = 84.0, b = 97.5, c = 108.1 A, beta = 93.0 degrees . Assuming the presence of two protomers per crystallographic asymmetric unit, the Matthews coefficient V(M) was calculated to be 3.5 A(3) Da(-1) and the solvent content was 65%.

Cooperativity of two active sites in bacterial homodimeric aconitases.

Aconitase catalyzes a reversible isomerization of citrate into isocitrate in the Krebs cycle. Escherichia coli possesses two kinds of aconitases, aconitase A (AcnA) and B (AcnB), whose structural organizations are different. We analyzed the structural state of AcnA by the chemical crosslinking and small-angle X-ray scattering. The protein adopts a homodimer in solution, as AcnB does. The catalytic assay of the two aconitases revealed that the isomerization of isocitrate displayed a negative cooperativity of the two active sites within each homodimer. On the other hand, insignificant cooperativity was observed in the reverse reaction. Therefore, the homodimerization of AcnAB yields a substrate-dependent cooperative effect. In conjunction with the dissociable homodimer of AcnB, the catalytic property could affect the intracellular metabolic process involving the Krebs cycle.

Versatile architecture of a bacterial aconitase B and its catalytic performance in the sequential reaction coupled with isocitrate dehydrogenase.

Aconitase B (AcnB) and isocitrate dehydrogenase (ICDH) catalyze the sequential reaction in the Krebs cycle. Since each enzyme was characterized as an independent protein in a diluted condition, the catalytic performance within the cellular metabolism remains unclear. In particular, high macromolecular concentration in the cytosol promotes weak interactions, which affects structure and function of the proteins. We found that the two bacterial enzymes exhibit variable catalytic performance of the sequential reaction, depending on the oligomerization state. The small-angle solution X-ray scattering and the chemical crosslinking analyses revealed that not only the two enzymes but also the fusion protein of the two enzymes assume homodimers in solution. Interestingly, the fusion protein maintains the homodimeric architecture of ICDH, but not AcnB. Instead, one of the two monomeric AcnB regions associates with the homodimeric ICDH region. The fusion protein displayed different catalytic performance of the sequential reaction from that observed in the mixture of the AcnB and ICDH proteins in an equimolar ratio. Connecting the two proteins by a flexible linker yielded a locally high concentration to promote the weak protein-protein interaction. The versatile architecture of AcnB may alter the metabolic process involving the Krebs cycle.

Expression, purification, crystallization and preliminary X-ray analysis of the ligand-binding domain of metabotropic glutamate receptor 7.

Glutamate is the major excitatory neurotransmitter and its metabotropic glutamate receptor (mGluR) plays an important role in the central nervous system. The ligand-binding domain (LBD) of mGluR subtype 7 (mGluR7) was produced using the baculovirus expression system and purified from the culture medium. The purified protein was characterized by gel-filtration chromatography, SDS-PAGE and a ligand-binding assay. Crystals of mGluR7 LBD were grown at 293 K by the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to 3.30 A resolution using synchrotron radiation and belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 92.4, c = 114.3 A. Assuming the presence of one protomer per crystallographic asymmetric unit, the Matthews coefficient V(M) was calculated to be 2.5 A3 Da(-1) and the solvent content was 51%.

Structures of the extracellular regions of the group II/III metabotropic glutamate receptors.

Metabotropic glutamate receptors play major roles in the activation of excitatory synapses in the central nerve system. We determined the crystal structure of the entire extracellular region of the group II receptor and that of the ligand-binding region of the group III receptor. A comparison among groups I, II, and III provides the structural basis that could account for the discrimination of group-specific agonists. Furthermore, the structure of group II includes the cysteine-rich domain, which is tightly linked to the ligand-binding domain by a disulfide bridge, suggesting a potential role in transmitting a ligand-induced conformational change into the downstream transmembrane region. The structure also reveals the lateral interaction between the two cysteine-rich domains, which could stimulate clustering of the dimeric receptors on the cell surface. We propose a general activation mechanism of the dimeric receptor coupled with both ligand-binding and interprotomer rearrangements.

Structure of the whole cytosolic region of ATP-dependent protease FtsH.

An ATP-dependent protease, FtsH, digests misassembled membrane proteins in order to maintain membrane integrity and digests short-lived soluble proteins in order to control their cellular regulation. This enzyme has an N-terminal transmembrane segment and a C-terminal cytosolic region consisting of an AAA+ ATPase domain and a protease domain. Here we present two crystal structures: the protease domain and the whole cytosolic region. The cytosolic region fully retains an ATP-dependent protease activity and adopts a three-fold-symmetric hexameric structure. The protease domains displayed a six-fold symmetry, while the AAA+ domains, each containing ADP, alternate two orientations relative to the protease domain, making "open" and "closed" interdomain contacts. Apparently, ATPase is active only in the closed form, and protease operates in the open form. The protease catalytic sites are accessible only through a tunnel following from the AAA+ domain of the adjacent subunit, raising a possibility of translocation of polypeptide substrate to the protease sites through this tunnel.

[Trends in the incidence of stroke in Yamagata, Japan].

The purpose of this study was to clarify the incidence and features of acute cerebrovascular diseases in Yamagata Prefecture, by comparing with those in other prefectures reported in previous studies. Consecutive 13,639 cases of acute stroke were prospectively registered to Yamagata Society in Treatment for Cerebral Stroke (YSTCS) between January 1, 1998 and December 31, 2002. Cerebral infarction (CI), intracerebral hemorrhage (CH), subarachnoid hemorrhage (SAH) and transient ischemic attack (TIA) were observed in 58.4%, 25.8%, 11.4% and 4.4% of the patients, respectively. The frequencies of CH and SAH (37.2%) in Yamagata Prefecture were higher than those reported in other studies (p < 0.01). In addition, time from onset to admission in Yamagata Prefecture was longer than that reported in others(p< 0.01). This study was one of the largest stroke registration studies in Japan enrolling 13,639 patients. In addition, computed tomography (CT) and/or magnetic resonance image (MRI) was performed on admission in all patients. Diagnosis was made by a neurologist or a neurosurgeon based on CT and/or MRI findings. Our results might accurately reflect current status of stroke patients in Yamagata Prefecture.

Stoichiometric complex formation by proliferating cell nuclear antigen (PCNA) and its interacting protein: purification and crystallization of the DNA polymerase and PCNA monomer mutant complex from Pyrococcus furiosus.

Replicative DNA polymerase interacts with processivity factors, the beta-subunit of DNA polymerase III or proliferating cell nuclear antigen (PCNA), in order to function with a long template DNA. The archaeal replicative DNA polymerase from Pyrococcus furiosus interacts with PCNA via its PCNA-interacting protein (PIP) motif at the C-terminus. The PCNA homotrimeric ring contains one PIP interacting site on each monomer and since the ring can accommodate up to three molecules simultaneously, formation of a stable stoichiometric complex of PCNA with its interacting protein has been difficult to control in vitro. A stable complex of the DNA polymerase with PCNA, using a PCNA monomer mutant, has been purified and crystallized. The best ordered crystal diffracted to 3.0 A resolution using synchrotron radiation. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 A.

Ligand-induced domain rearrangement of fatty acid beta-oxidation multienzyme complex.

The quaternary structure of a fatty acid beta-oxidation multienzyme complex, catalyzing three sequential reactions, was investigated by X-ray crystallographic and small-angle X-ray solution scattering analyses. X-ray crystallography revealed an intermediate structure of the complex among the previously reported structures. However, the theoretical scattering curves calculated from the crystal structures remarkably disagree with the experimental profiles. Instead, an ensemble of the atomic models, which were all calculated by rigid-body optimization, reasonably explained the experimental data. These structures significantly differ from those in the crystals, but they maintain the substrate binding pocket at the domain boundary. Comparisons among these structures indicated that binding of 3-hydroxyhexadecanoyl-CoA or nicotinamide adenine dinucleotide induces domain rearrangements in the complex. The conformational changes suggest the structural events occurring during the chain reaction catalyzed by the multienzyme complex.

Crystal structure of human lectin-like, oxidized low-density lipoprotein receptor 1 ligand binding domain and its ligand recognition mode to OxLDL.

Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, is the major receptor for oxidized LDL (OxLDL) in endothelial cells. We have determined the crystal structure of the ligand binding domain of LOX-1, with a short stalk region connecting the domain to the membrane-spanning region, as a homodimer linked by an interchain disulfide bond. In vivo assays with LOX-1 mutants revealed that the "basic spine," consisting of linearly aligned arginine residues spanning over the dimer surface, is responsible for ligand binding. Single amino acid substitution in the dimer interface caused a severe reduction in LOX-1 binding activity, suggesting that the correct dimer arrangement is crucial for binding to OxLDL. Based on the LDL model structure, possible binding modes of LOX-1 to OxLDL are proposed.

Crystal structure and functional implications of Pyrococcus furiosus hef helicase domain involved in branched DNA processing.

DNA and RNA frequently form various branched intermediates that are important for the transmission of genetic information. Helicases play pivotal roles in the processing of these transient intermediates during nucleic acid metabolism. The archaeal Hef helicase/ nuclease is a representative protein that processes flap- or fork-DNA structures, and, intriguingly, its C-terminal half belongs to the XPF/Mus81 nuclease family. Here, we report the crystal structure of the helicase domain of the Hef protein from Pyrococcus furiosus. The structure reveals a novel helical insertion between the two conserved helicase core domains. This positively charged extra region, structurally similar to the "thumb" domain of DNA polymerase, plays critical roles in fork recognition. The Hef helicase/nuclease exhibits sequence similarity to the Mph1 helicase from Saccharomyces cerevisiae; XPF/Rad1, involved in DNA repair; and a putative Hef homolog identified in mammals. Hence, our findings provide a structural basis for the functional mechanisms of this helicase/nuclease family.

Overexpression, purification and crystallization of an archaeal DNA ligase from Pyrococcus furiosus.

DNA ligases seal single-strand breaks in double-stranded DNA and their function is essential to maintain the integrity of the genome during various aspects of DNA metabolism, such as replication, excision repair and recombination. DNA-strand breaks are frequently generated as reaction intermediates in these events and the sealing of these breaks depends solely on the proper function of DNA ligase. Crystals of the archaeal DNA ligase from Pyrococcus furiosus were obtained using 6.6%(v/v) ethanol as a precipitant and diffracted X-rays to 1.7 A resolution. They belong to the monoclinic space group P2(1), with unit-cell parameters a = 61.1, b = 88.3, c = 63.4 A, beta = 108.9 degrees. The asymmetric unit contains one ligase molecule.

Structural basis for channelling mechanism of a fatty acid beta-oxidation multienzyme complex.

The atomic view of the active site coupling termed channelling is a major subject in molecular biology. We have determined two distinct crystal structures of the bacterial multienzyme complex that catalyzes the last three sequential reactions in the fatty acid beta-oxidation cycle. The alpha2beta2 heterotetrameric structure shows the uneven ring architecture, where all the catalytic centers of 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD) and 3-ketoacyl-CoA thiolase (KACT) face a large inner solvent region. The substrate, anchored through the 3'-phosphate ADP moiety, allows the fatty acid tail to pivot from the ECH to HACD active sites, and finally to the KACT active site. Coupling with striking domain rearrangements, the incorporation of the tail into the KACT cavity and the relocation of 3'-phosphate ADP bring the reactive C2-C3 bond to the correct position for cleavage. The alpha-helical linker specific for the multienzyme contributes to the pivoting center formation and the substrate transfer through its deformation. This channelling mechanism could be applied to other beta-oxidation multienzymes, as revealed from the homology model of the human mitochondrial trifunctional enzyme complex.

Negative cooperativity of glutamate binding in the dimeric metabotropic glutamate receptor subtype 1.

Metabotropic glutamate receptor (mGluR) subtype 1 is a Class III G-protein-coupled receptor that is mainly expressed on the post-synaptic membrane of neuronal cells. The receptor has a large N-terminal extracellular ligand binding domain that forms a homodimer, however, the intersubunit communication of ligand binding in the dimer remains unknown. Here, using the intrinsic tryptophan fluorescence change as a probe for ligand binding events, we examined whether allosteric properties exist in the dimeric ligand binding domain of the receptor. The indole ring of the tryptophan 110, which resides on the upper surface of the ligand binding pocket, sensed the ligand binding events. From saturation binding curves, we have determined the apparent dissociation constants (K(0.5)) of representative agonists and antagonists for this receptor (3.8, 0.46, 40, and 0.89 microm for glutamate, quisqualate, (S)-alpha-methyl-4-carboxyphenylglycine ((S)-MCPG), and (+)-2-methyl-4-carboxyphenylglycine (LY367385), respectively). Calcium ions functioned as a positive modulator for agonist but not for antagonist binding (K(0.5) values were 1.3, 0.21, 59, and 1.2 microm for glutamate, quisqualate, (S)-MCPG, and LY367385, respectively, in the presence of 2.0 mm calcium ion). Moreover, a Hill analysis of the saturation binding curves revealed the strong negative cooperativity of glutamate binding between each subunit in the dimeric ligand binding domain. As far as we know, this is the first direct evidence that the dimeric ligand binding domain of mGluR exhibits intersubunit cooperativity of ligand binding.

Overexpression of rat heat shock protein 70 reduces neuronal injury after transient focal ischemia, transient global ischemia, or kainic acid-induced seizures.

OBJECTIVE: Transgenic (Tg) mice overexpressing rat heat shock protein 70 (hsp70) demonstrated less infarction than did wild-type (WT) littermates after permanent focal cerebral ischemia. The purpose of this study was to determine whether neuronal injury and apoptosis were reduced in hsp70 Tg mice after transient focal ischemia. The effects of hsp70 overexpression were also evaluated after transient global ischemia or kainic acid (KA)-induced seizures, to verify the results in other excitotoxic stress models. METHODS: Transient focal ischemia was produced with middle cerebral artery occlusion via intraluminal suture cannulation. Infarction volumes were assessed 24 hours after 30 minutes of middle cerebral artery occlusion. Transient global ischemia was produced with 25 minutes of bilateral common carotid artery occlusion. KA (30 mg/kg) was administered subcutaneously, and seizure activity was evaluated. The number of eosinophilic neurons was assessed in the CA1 region 72 hours after bilateral common carotid artery occlusion and in the CA3 region 24 hours after KA administration. RESULTS: The infarction volume after transient middle cerebral artery occlusion was significantly smaller in hsp70 Tg mice than in WT mice (9.1 +/- 5.7 mm(3) versus 22.4 +/- 16.8 mm(3), P < 0.05). The number of eosinophilic neurons in the CA1 area after bilateral common carotid artery occlusion and in CA3 after KA injection was significantly lower in hsp70 Tg mice than in WT mice (949.1 +/- 1095.5 versus 2406.9 +/- 1380.3, P < 0.05, and 33.8 +/- 45.3 versus 119.4 +/- 112.1, P < 0.05, respectively). Fewer terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling-positive cells were observed in hsp70 Tg mice than in WT mice in each model. CONCLUSION: The results demonstrate that overexpression of hsp70 reduces neuronal injury after ischemia and seizures. The reduction in the number of terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling-positive cells in hsp70 Tg mice suggests that hsp70 overexpression might reduce apoptotic cell death.