An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p.

Ykt6p is a nonsyntaxin SNARE implicated in multiple intracellular membrane trafficking steps. Here we present the structure of the NH2-terminal domain of Ykt6p (Ykt6pN, residues 1 to 140). The structure of Ykt6pN differed entirely from that of syntaxin and resembled the overall fold of the actin regulatory protein, profilin. Like some syntaxins, Ykt6p adopted a folded back conformation in which Ykt6pN bound to its COOH-terminal core domain. The NH2-terminal domain plays an important biological role in the function of Ykt6p, which in vitro studies revealed to include influencing the kinetics and proper assembly of SNARE complexes.

Structural basis of diverse sequence-dependent target recognition by the 8 kDa dynein light chain.

Dyneins are multi-subunit molecular motors that translocate molecular cargoes along microtubules. Other than acting as an essential component of the dynein motor complex, the 89-residue subunit of dynein light chain (DLC8) also regulates a number of other biological events by binding to various proteins and enzymes. Currently known DLC8 targets include neuronal nitric oxide synthase; the proapoptotic Bcl-2 family member protein designated Bim; a Drosophila RNA localization protein Swallow, myosin V, neuronal scaffolding protein GKAP, and IkappaBalpha, an inhibitor of the NFkappaB transcription factor. The DLC8-binding domains of the various targets are confined within a short, continuous stretch of amino acid residues. However, these domains do not share any obvious sequence homology with each other. Here, the three-dimensional structures of DLC8 complexed with two peptides corresponding to the DLC8-binding domains of neuronal nitric oxide synthase and Bim, respectively, were determined by NMR spectroscopy. Although the two DLC8-binding peptides have entirely different amino acid sequences, both peptides bind to the protein with a remarkable similar conformation by engaging the symmetric DLC8 dimer through antiparallel beta-sheet augmentation via the beta2 strand of the protein. Structural comparison indicates that the two target peptides use different regions within the conformational flexible peptide-binding channels to achieve binding specificity. We have also re-determined the apo-form solution structure of DLC8 in this work. The structures of the DLC8/target peptide complexes, together with the dynamic properties of the protein, provide a molecular basis of DLC8's diverse amino acid sequence-dependent target recognition.

Solution structure and backbone dynamics of the second PDZ domain of postsynaptic density-95.

The second PDZ domain of postsynaptic density-95 (PSD-95 PDZ2) plays a critical role in coupling N-methyl-D-aspartate receptors to neuronal nitric oxide synthase (nNOS). In this work, the solution structure of PSD-95 PDZ2 was determined to high resolution by NMR spectroscopy. The structure of PSD-95 PDZ2 was compared in detail with that of alpha1-syntrophin PDZ domain, as the PDZ domains share similar target interaction properties. The interaction of the PSD-95 PDZ2 with a carboxyl-terminal peptide derived from a cytoplasmic protein CAPON was studied by NMR titration experiments. Complex formation between PSD-95 PDZ2 and the nNOS PDZ was modelled on the basis of the crystal structure of the alpha1-syntrophin PDZ/nNOS PDZ dimer. We found that the prolonged loop connecting the betaB and betaC strands of PSD-95 PDZ2 is likely to play a role in both the binding of the carboxyl-terminal peptide and the nNOS beta-finger. Finally, the backbone dynamics of the PSD-95 PDZ2 in the absence of bound peptide were studied using a model-free approach. The "GLGF"-loop and the loop connecting alphaB and betaF of the protein display some degree of flexibility in solution. The rest of the protein is rigid and lacks detectable slow time-scale (microseconds to milliseconds) motions. In particular, the loop connecting betaB and betaC loop adopts a well-defined, rigid structure in solution. It appears that the loop adopts a pre-aligned conformation for the PDZ domain to interact with its targets.

Formation of nNOS/PSD-95 PDZ dimer requires a preformed beta-finger structure from the nNOS PDZ domain.

PDZ domains are modular protein units that play important roles in organizing signal transduction complexes. PDZ domains mediate interactions with both C-terminal peptide ligands and other PDZ domains. Here, we used PDZ domains from neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) to explore the mechanism for PDZ-dimer formation. The nNOS PDZ domain terminates with a approximately 30 residue amino acid beta-finger peptide that is shown to be required for nNOS/PSD-95 PDZ dimer formation. In addition, formation of the PDZ dimer requires this beta-finger peptide to be physically anchored to the main body of the canonical nNOS PDZ domain. A buried salt bridge between the beta-finger and the PDZ domain induces and stabilizes the beta-hairpin structure of the nNOS PDZ domain. In apo-nNOS, the beta-finger peptide is partially flexible and adopts a transient beta-strand like structure that is stabilized upon PDZ dimer formation. The flexibility of the NOS PDZ beta-finger is likely to play a critical role in supporting the formation of nNOS/PSD-95 complex. The experimental data also suggest that nNOS PDZ and the second PDZ domain of PSD-95 form a "head-to-tail" dimer similar to the nNOS/syntrophin complex characterized by X-ray crystallography.

Cytokine-inducing glycolipids in the lipoteichoic acid fraction from Enterococcus hirae ATCC 9790.

Five high molecular weight glycolipids capable of stimulating human peripheral whole-blood cell cultures to cause interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha induction were isolated from one of the lipoteichoic acid fractions (LTA-2) extracted from Enterococcus hirae ATCC 9790 (Tsutsui et al., (1991) FEMS Microbiol. Immunol. 76, 211-218) by a combination of hydrophobic interaction and anion-exchange chromatographies. This purification procedure resulted in a remarkable increase in the cytokine-inducing activities on the weight basis of isolated glycolipids (a maximum of 36- and 17-fold increases of IL-6 and TNF-alpha induction, respectively). The total yield of these bioactive glycolipids amounted to 6 wt% of the parent LTA-2 fraction, while the recovery rate in terms of the cytokine-inducing activities was estimated to be sufficient. The chemical composition and the profile, using SDS-PAGE, revealed that all of the isolated bioactive components were high molecular weight glycolipids, which were distinct from each other and from the parent LTA-2 fraction. These findings suggest that the IL-6 and TNF-alpha-inducing activities previously noted in the parent LTA-2 fraction are not attributable to a chemical entity, the structure of which had been proposed elsewhere (Fischer, W. (1990) in Glycolipids, Phosphoglycolipids and Sulfoglycolipids (Kates, M. ed.) pp. 123-234, Plenum Press, New York), but to the other high molecular weight glycolipids described here.

Chondroma of the spine in a newborn infant: case report.

We report a newborn infant with a chondroma of the spine covered by a large lipoma. The child had no neurological deficit. Presenting as a tumor of the back, the chondroma was successfully excised. A review of the pertinent literature emphasizes the rarity of this lesion.

Intratumoral hemorrhage after a ventriculoperitoneal shunting procedure.

Ventriculoperitoneal shunting has been accepted as a safe and useful preliminary procedure that lowers the mortality and morbidity of definitive surgery for tumors causing obstructive hydrocephalus. We are reporting four patients with intratumoral hemorrhage as a complication of shunting. The hemorrhage was massive and fatal in two patients, one with an unverified pineal tumor and the other with a malignant astrocytoma of the thalamus. The hemorrhage was small and limited in the other two patients, one with a glioblastoma of the thalamus and the other with a cerebellar astrocytoma. On the basis of this experience, we conclude that the possibility of intratumoral hemorrhage should be taken into consideration when planning the preoperative management of obstructive hydrocephalus caused by brain tumors. It is possible that ventricular decompression may result in rapid motion and distortion of the intracranial structures and a sudden imbalance between intracranial and intratumoral pressures, leading to vascular insufficiency, congestion, and then hemorrhage within the tumor.

Spontaneous resolution of chronic epidural hematomas: report of three cases.

We report three cases of the spontaneous resolution of a traumatic chronic epidural hematoma. The patients were young adults with only mild headache after trauma. All patients were treated conservatively because of the very mild symptoms and the time lapse of 1 to 2 weeks between trauma and our unexpected detection of the frontal epidural hematomas. Slow, spontaneous resolution of the hematoma occurred in all three cases.

The effect of atherosclerosis on endothelium-dependent relaxation in the aorta and intracranial arteries of rabbits.

The effect of hypercholesterolemia on vascular responsiveness was studied in isolated rabbit arteries. The arteries of animals maintained on a cholesterol-rich (1%) diet for 6 months had more pronounced intimal lesion than those receiving the diet for 3 months. The aortas were more severely damaged than the carotid or basilar arteries. Segments of the arteries were mounted in organ chambers for isometric tension recording or for measurement of the endothelium-derived relaxing factor. Endothelium-independent relaxation induced by glyceryl trinitrate was not affected even in the most severely damaged arteries; endothelium-dependent relaxation in response to acetylcholine (ACh) and A23187 was progressively inhibited as the degree of fatty streak formation increased. In the carotid arteries, mean (+/- standard deviation) relaxation induced by 10(-5) M of ACh (expressed as a percentage of the maximum relaxation induced by 10(-4) M of papaverine) decreased from 87.33% +/- 6.30% in control tissues to 60.90% +/- 4.64% in vessels from animals subjected to 6 months of hypercholesterolemia (p less than 0.01); in the aortas, mean relaxation due to 10(-5) M of ACh was 85.08% +/- 8.03% in control tissues and 41.35% +/- 13.68% in hypercholesterolemic tissues (p less than 0.01). In the carotid arteries, mean relaxation induced by 10(-7) M of A23187 decreased from 95.81% +/- 3.58% in control tissues to 55.95% +/- 2.81% in hypercholesterolemic tissues (p less than 0.01); in the aortas, relaxation in response to 10(-7) M of A23187 was 73.73% +/- 4.35% in control tissues and 29.35% +/- 6.77% in hypercholesterolemic tissues (p less than 0.01). Intimal lesions were not produced in the basilar arteries even in rabbits with 12 months of hypercholesterolemia, and endothelium-dependent relaxation was preserved.

Intermolecular contacts between the lambda-Cro repressor and the operator DNA characterized by nuclear magnetic resonance spectroscopy.

The specific interaction between lambda phage Cro repressor and the DNA fragment bearing the consensus sequence of operators has been studied using nuclear magnetic resonance (NMR). Using both 15N- and 13C/15N- labeled lambda-Cro in complex with unlabeled DNA, chemical shift assignments of the lambda-Cro-DNA complex were obtained using heteronuclear NMR experiments. Inter-molecular contacts between the protein and DNA were identified using heteronuclear filtered NOESY experiments. The inter-molecular contacts were supplemented with intra-protein and intra-DNA NOE constraints to dock lambda-Cro to the bent B-form DNA using restrained molecular dynamics. The structure of one of the subunits of lambda-Cro in the complex is essentially the same as that of the unbound form. In the complex, inter-molecular NOEs were observed between the "helix-turn-helix" region comprising the alpha2 and alpha3 helices of the lambda-Cro protein and the major groove of the DNA. The methyl group of Thr17 forms a hydrophobic contact with the methyl group of the thymine at base pair 1 in the DNA, and Val25 and Ala29 make hydrophobic contacts with the methyl group of the thymine at base pair 5. The presence and the absence of these contacts can explain the difference in the affinity of lambda-Cro to several variants of the operator sequence.

Intracranial aneurysm associated with moyamoya disease in childhood.

We report a 6-year-old girl with an intracranial aneurysm associated with moyamoya disease. The patient did not have a subarachnoid hemorrhage but had an ischemic attack. The aneurysm, located on the proximal portion of the lateral posterior choroidal artery, disappeared after external carotid-internal carotid anastomosis. The characteristics of the aneurysms associated with moyamoya disease are briefly reviewed. We emphasize that the treatment of choice in moyamoya disease is cerebral revascularization, because it reduces the increased blood flow through the moyamoya vessels, basilar artery and other uninvolved cerebral arteries, the sites where the aneurysms in this disease frequently develop, as the blood flow through the external carotid system is increased by such an operation.

Traumatic cerebral swelling developing within 30 minutes after injury.

Development of acute massive cerebral swelling within 30 minutes after closed head injury is documented by computed tomography. The swelling was rapidly resolved by steroid treatment. Detection and treatment of acute traumatic cerebral swelling are discussed briefly.

Chondrosarcoma of the ethmoid sinus extending to the anterior fossa.

A 27-year-old women with chondrosarcoma of the ethmoid sinus extending to the anterior fossa had initial symptoms of blurred vision on the left and slight proptosis. Neuroradiological examination, however, disclosed that the tumor extended not only to the anterior cranial fossa, but also to the orbit, nasal cavity, and maxillary sinus. It is difficult to predict the biological behavior of chondrosarcoma by means of histological examination. The effectiveness of radiotherapy, chemotherapy, or both is also a matter of controversy. Clinical aspects of chondrosarcoma are briefly discussed.

Hypertensive putaminal hemorrhage: analysis of 182 patients.

The results of treatment for 182 patients with hypertensive putaminal hemorrhage are described. The patients are graded according to the level of consciousness on admission; grade 1, fully conscious; grade 2, somnolent; grade 3, stuporous; grade 4, semicomatose; and grade 5, deeply comatose. Removal of hemorrhage was performed in 44 patients (24%), and conservative treatment was given in 138 patients (76%). One-month mortality in the surgical group was 27%; it was 22% in the conservative group. At 6 months after surgical treatment, the percentages of patients who returned to full-time work or independent life without disability or with minimal disability (good recovery) were 40% in grade 1, 30% in grade 2, 22% in grade 3, 11% in grade 4, and 0% in grade 5; after conservative treatment, percentages with good recovery were 77% in grade 1, 56% in grade 2, 29% in grade 3, 7% in grade 4, and 0% in grade 5. This study shows that surgical treatment does not give better results in the management of hypertensive putaminal hemorrhage.

Accumulation of Tc-99m methylene diphosphonate in calcified metastatic lesions of the liver from colonic carcinoma. Comparison with calcification on X-ray computed tomogram.

Abnormal accumulation of Tc-99m MDP in two metastatic lesions of the liver was observed in a patient with resected colon carcinoma. Single-photon emission computed tomography (SPECT) revealed characteristic marginal accumulation of Tc-99m MDP in both of those metastatic lesions. X-ray CT showed the corresponding marginal calcification in one of the metastases, but no apparent calcification was observed in the other lesion. Two months later, however, the latter also became calcified on x-ray CT. These findings suggest that the accumulation of Tc-99m MDP in the present case is strongly related to the calcium deposition and that Tc-99m MDP may accumulate in a calcified metastatic lesion before the calcification appears on x-ray CT.

Chronic encapsulated intracerebral hematoma associated with angiographically occult arteriovenous malformation--case report.

A 34-year-old female presented with a rare chronic encapsulated intracerebral hematoma associated with an angiographically occult arteriovenous malformation. A neovascularized fibrous capsule containing various stages of intracerebral hematoma formation was removed. These unusual entities mimic brain tumors or abscesses because of gradual growth and slowly progressive neurological deficits. Repeated bleeding or exudation from the capillaries of the capsule may allow expansion of the chronic encapsulated intracerebral hematoma.

[Immunological examination in patients with brain tumor and the usefulness of PHA skin test (author's transl)].

The usefulness of in vivo skin test reaction to phytohemagglutinin to assess cell-mediated immunity was investigated in 109 subjects, including 37 normal controls, 35 patients without brain tumor and 37 patients with brain tumor. Intradermal injection at forearm of 10 microgram of Bacto-phytohemagglutinin-P (PHA-P) produced an erythema within 24 hours. Upon PHA skin reaction the mean diameter of erythema was 25.3 mm with the normal in the control and 18.6 mm with other diseases than brain tumor, while it was 13.0 mm with brain tumor. Furthermore when mean diameter of those with longer than 15.0 mm are classified as positive in the PHA skin test, the normal in the control revealed 89.2% positive and the patients with brain tumor indicated only 37.8% positive. A positive reaction was observed in 10 (77.0%) of 13 benign brain tumor and 5 (26.3%) of 19 malignant brain tumor. A correlation was found between in vivo and in vitro responses to PHA, and between PHA and PPD skin reaction. In repeated PHA skin test at intervals of about one month, the skin reaction to PHA of patients with brain tumor varied depending on the clinical states. Other advantages of this test are no presensitization and it can be used repeatedly. The PHA skin test is a useful and simple screening test for cellular immune function of patients with brain tumor and as a simple method applicable clinically indicating accurate immuno-reactivity the PHA skin tests have been carried out during the course of treatment with an interval of about one month repeatedly.