Structure of macrophage migration inhibitory factor in complex with methotrexate.

Methotrexate (MTX) is an anticancer and anti-rheumatoid arthritis drug that is considered to block nucleotide synthesis and the cell cycle mainly by inhibiting the activity of dihydrofolate reductase (DHFR). Using affinity-matrix technology and X-ray analysis, the present study shows that MTX also interacts with macrophage migration inhibitory factor (MIF). Fragment molecular-orbital calculations quantified the interaction between MTX and MIF based on the structure of the complex and revealed the amino acids that are effective in the interaction of MTX and MIF. It should be possible to design new small-molecule compounds that have strong inhibitory activity towards both MIF and DHFR by structure-based drug discovery.

Primary Intracranial Neuroendocrine Tumor of the Skull Base Complicated with Tension Pneumocephalus after Radiotherapy.

Neuroendocrine tumors (NETs) are neoplasms that originate from cells of the endocrine and nervous systems, and are commonly found in the gastrointestinal and respiratory tracts. Primary intracranial NETs are extremely rare and have been the focus of only a few studies thus far. Herein, we report the case of a primary intracranial NET of the skull base complicated with tension pneumocephalus after radiotherapy. An 84-year-old woman visited a local hospital for a head injury, and CT revealed a skull base tumor. MRI showed that the tumor was located mainly on the clivus and extended into the paranasal sinuses and nasal cavity. We biopsied the tumor via the nasal cavity, and the pathological diagnosis was NET, WHO grade 2. We subsequently administered focal intensity-modulated radiation therapy, but the patient developed tension pneumocephalus 1 year after radiotherapy. We therefore performed endoscopic transnasal cerebrospinal fluid leak closure with a nasoseptal flap. The postoperative course was successful, and the patient returned home but died of an unknown cause 2 years after discharge. The optimal postoperative management of primary intracranial NETs remains controversial. Tension pneumocephalus related to radiotherapy is a rare complication. Assessing skull bone erosion before radiotherapy and performing regular radiological follow-up examinations are essential to prevent this rare complication.

Mucinous myoepithelioma: A report of a new variant.

Myoepitheliomas account for approximately 1.5% of all salivary gland tumors and arise most frequently from the parotid gland. Recently, a new myoepithelioma variant, called mucinous myoepithelioma, has attracted widespread attention. These tumors are recognized as a unique subtype of myoepithelioma, characterized by the presence of abundant mucin. We herein report the findings of an 86-year-old Japanese woman who presented with a hard mass of the right parotid gland behind her right ear which was gradually increasing in size. The patient had undergone a fine-needle aspiration biopsy 4 years earlier, and a cytological evaluation of a biopsy specimen had shown features of pleomorphic adenoma. A resection was thus performed and the tissue was found to be an encapsulated, soft and solid mass, and the cut surface was observed to be a capsulated and well-defined tumor lesion with myxoid-looking foci of gray-white coloration. Microscopic examination revealed that this lesion was composed of a proliferation of bland-looking epithelial and myoepithelial cells, arranged in a solid or reticular growth fashion in an abundant myxomatous or hyalinized stroma. These neoplastic epithelial cells had centrally located small nuclei with fine chromatin and abundant clear to eosinophilic cytoplasm, often containing mucin in a uniform pattern. Immunohistochemical staining demonstrated the tumor cells to be positive for AE1/AE3, S-100 and mucicarmine. Our findings suggest this case to be one myoepithelioma variant of mucinous myoepithelioma, and more experience related to this myoepithelioma variant is necessary to better understand its biological behavior and make an accurate diagnosis for a proper treatment.

Total Synthesis and Biological Evaluation of 19-Hydroxysarmentogenin-3 -O-α-l-rhamnoside, Trewianin, and Their Aglycons.

Cardenolides comprise an important family of natural steroids with a wide spectrum of biological activities. Although 19-hydroxysarmentogenin-3- O-α-l-rhamnoside (1a) and trewianin (1b) were structurally determined to have cardenolide structures, their biological activities have not been evaluated. The 6/6/6/5-membered ABCD-ring systems of both 1a and 1b are decorated by a ß-oriented C17-butenolide, three C11,14,19-hydroxy groups, and a C3-O-l-rhamnoside moiety. On the other hand, 1a and 1b are epimeric at the C5-position. The structures of 1a and 1b were assembled from four simple fragments by applying a convergent and unified strategy. The AB-ring 10a/b and the D-ring 8/9 were tentatively tethered at the acetal moiety, and a subsequent stereoselective 6- exo radical reaction linked the two fragments. Next, an aldol reaction enabled simultaneous introduction of three new stereocenters of the C-rings of 5aa and 54. Attachment of the C17-butenolide led to aglycons 2a and 2b. l-Rhamnose was then installed into 2a and 2b to yield the targets 1a and 1b, respectively. Finally, the growth inhibitory activity of 1a, 1b, 2a, and 2b was assessed against MCF-7 human breast carcinoma cells. The significantly higher activities of 1a and 1b in comparison to 2a and 2b demonstrated the biological importance of the monosaccharide substructure.

Studies on Instructive Construction of exo-Olefin Terminated Five- and Six-Membered Nitrogen Heterocycles: SmI2-Mediated Intramolecular Cyclization of Haloalkynals.

Stereoselective construction of exo-olefin terminated pyrrolidine and piperidine frameworks was developed by employing SmI2-mediated intramolecular radical cyclization of haloalkynaks. The radical cyclization affording 2,3-disubstituted pyrrolidines and piperidines proceeded in a highly stereoselective manner. However, decreasing stereoselectivety was observed in the preparation of 2,4-disubstituted pyrrolidine and 3,4-disubstituted piperidine derivatives in the cyclization.

Myoepithelial carcinoma of the parotid gland: A case of adequate fine-needle aspiration cytology specimens rendering a conclusive diagnosis possible.

An 80-year-old male presented with a history of a hard right parotid mass that had gradually increased in size, with subsequent facial paralysis. A fine-needle aspiration biopsy was performed. The cytologic specimens contained a substantial number of sheet-like clusters or small groups of a mixture of plasmacytoid, oval to spindled, or large epithelioid cells having hyperchromatic pleomorphic nuclei, abundant cytoplasm with occasional inclusion body-like materials, and prominent nucleoli, in a relatively clear background. We first interpreted it as a carcinoma, suggestive of myoepithelial differentiation. Radical parotidectomy was performed, and a gross examination of the neoplasm revealed a non-capsulated and ill-defined tumor lesion, with a grayish or yellowish cut surface, associated with fat invasion. On a microscopic examination, the tumor was predominantly composed of the solid proliferation of atypical cells including a mixture of oval to spindled, plasmacytoid, or epithelioid cells, often arranged in a trabecular and reticular growth pattern with patchy eosinophilic hyalinized stroma. Immunohistochemistry showed that the carcinoma cells were specifically positive for p63, cytokeratins, and vimentin. Finally, electron microscopy demonstrated that their phenotype was consistent with a myoepithelial origin containing many bundles of variably thin actin filaments. Therefore, we finally made a diagnosis of myoepithelial carcinoma, defined as the malignant counterpart of benign myoepithelioma. We should be aware that owing to its characteristic cytological features, cytopathologists may be able to make a correct diagnosis of myoepithelial carcinoma, based on multiple and adequate samplings.

NEUROD1-deficient diabetes (MODY6): Identification of the first cases in Japanese and the clinical features.

AIMS: Only a few families with neuronal differentiation 1 (NEUROD1)-deficient diabetes, currently designated as maturity-onset diabetes of the young 6 (MODY6), have been reported, but mostly in Caucasian, and no mutation has been identified by family-based screening in Japanese. Accordingly, the phenotypic details of the disease remain to be elucidated. METHODS: We examined a total of 275 subjects having diabetes suspected to be MODY who were negative for mutations in MODY1-5 referred from 155 medical institutions throughout Japan. So as not to miss low penetrant cases, we examined non-obese Japanese patients with early-onset diabetes regardless of the presence of family history by direct sequencing of all exons and flanking regions of NEUROD1 . Large genomic rearrangements also were examined. RESULTS: Four patients with 3 frameshift mutations and 1 missense mutation, all of which were heterozygous and 3 of which were novel, were identified. Diabetic ketosis was found occasionally in these patients even under conditions of chronic hyperglycemia, for unknown reasons. Although the capacity of early-phase insulin secretion was low in these patients, the insulin secretory capacity was relatively preserved compared to that in hepatocyte nuclear factor (HNF)1A- and HNF1B-MODY. One of the patients and 2 of their diabetic mothers were found to have some mental or neuronal abnormality. CONCLUSIONS: This is the first report of NEUROD1 mutations in Japanese, who have a genetic background of intrinsically lower capacity of insulin secretion. NEUROD1-deficient diabetes appears to be low penetrant, and may occur in concert with other genetic factors.

Efficient ruthenium sensitizer with an extended π-conjugated terpyridine ligand for dye-sensitized solar cells.

A ruthenium sensitizer with an extended π-conjugated terpyridine (TUS-42) has been synthesized for dye-sensitized solar cells (DSCs). Upon extension of the π-conjugated system of the terpyridine ligand, the conversion efficiency of the DSC with TUS-42 improved successfully to 10.7%, which is almost comparable to that of one of the most efficient ruthenium sensitizers (Black dye). Interestingly, an extremely large short-circuit current density (Jsc) value (22.7 mA/cm(2)) was obtained in the DSC with TUS-42 even though the amount of dye adsorption to the TiO2 photoelectrode is relatively small.

Ruthenium sensitizers having an ortho-dicarboxyl group as an anchoring unit for dye-sensitized solar cells: synthesis, photo- and electrochemical properties, and adsorption behavior to the TiO2 surface.

A novel ruthenium sensitizer ((TBA)[Ru(3',4'-dicarboxyterpyridine)(NCS)3], TBA = tetrabutylammonium, TUS-28) has been synthesized as an improved model sensitizer for (TBA)[Ru(4'-(3,4-dicarboxyphenyl)terpyridine)(NCS)3] (TUS-20). The molar absorptivity of TUS-28 in the whole visible region was smaller than that of TUS-20 due to the absence of the phenyl ring at the terpyridine ligand. On the other hand, the energy levels of HOMO and LUMO of TUS-28 were still suitable for effective electron transfer reactions in dye-sensitized solar cells (DSCs). TUS-28 did not show a superior adsorptivity to the TiO2 surface just like TUS-20, even though TUS-28 has an ortho-dicarboxyl group which is reported to be an efficient anchoring unit. ATR-IR measurements revealed that both carboxyl groups of TUS-28 participate in binding to the TiO2 surface just like TUS-20. Therefore, steric hindrance between the hydrogen atom at the 4-position of 3',4'-dicarboxyterpyridine and the OH group at the TiO2 surface seems to retard the rapid adsorption, and to weaken the binding strength of TUS-28. The DSC with TUS-28 showed 8.2% conversion efficiency, which is higher than that of TUS-20 (7.5%). The J(sc) value of the DSC with TUS-28 was larger than that of TUS-20 even though the amount of dye adsorption of TUS-28 was smaller than that of TUS-20. A more effective electron injection reaction is considered to contribute mainly to the efficiency improvement of TUS-28 since the path length of the electron injection from TUS-28 to the conduction band of the TiO2 is shorter than that of TUS-20 due to the absence of the phenyl ring.

Structure of the ß-form of human MK2 in complex with the non-selective kinase inhibitor TEI-L03090.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAP-K2), a serine/threonine kinase from the p38 mitogen-activated protein kinase signalling pathway, plays an important role in the production of TNF-α and other cytokines. In a previous report, it was shown that MK2 in complex with the selective inhibitor TEI-I01800 adopts an α-helical glycine-rich loop that is induced by the stable nonplanar conformer of TEI-I01800. To understand the mechanism of the structural change, the structure of MK2 bound to TEI-L03090, which lacks the key substituent found in TEI-I01800, was determined. MK2-TEI-L03090 has a ß-sheet glycine-rich loop in common with other kinases, as predicted. This result suggests that a small compound can induce a drastic conformational change in the target protein structure and can be used to design potent and selective inhibitors.

Crystal structure of human cyclin-dependent kinase-2 complex with MK2 inhibitor TEI-I01800: insight into the selectivity.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAP-K2) is a Ser/Thr kinase from the p38 mitogen-activated protein kinase signalling pathway and plays an important role in inflammatory diseases. The crystal structure of the MK2-TEI-I01800 complex has been reported; its Gly-rich loop was found to form an α-helix, not a ß-sheet as has been observed for other Ser/Thr kinases. TEI-I01800 is 177-fold selective against MK2 compared with CDK2; in order to understand the inhibitory mechanism of TEI-I01800, the cyclin-dependent kinase 2 (CDK2) complex structure with TEI-I01800 was determined at 2.0 Å resolution. Interestingly, the Gly-rich loop of CDK2 formed a ß-sheet that was different from that of MK2. In MK2, TEI-I01800 changed the secondary structure of the Gly-rich loop from a ß-sheet to an α-helix by collision between Leu70 and a p-ethoxyphenyl group at the 7-position and bound to MK2. However, for CDK2, TEI-I01800 bound to CDK2 without this structural change and lost the interaction with the substituent at the 7-position. In summary, the results of this study suggest that the reason for the selectivity of TEI-I01800 is the favourable conformation of TEI-I01800 itself, making it suitable for binding to the α-form MK2.

Structure basis 1/2SLPI and porcine pancreas trypsin interaction.

SLPI (secretory leukocyte protease inhibitor) is a 107-residue protease inhibitor which inhibits various serine proteases, including elastase, cathepsin G, chymotrypsin and trypsin. SLPI is obtained as a multiple inhibitor in lung defense and in chronic airway infection. X-ray crystal structures have so far reported that they are full-length SLPIs with bovine α-chymotrypsin and 1/2SLPI (recombinant C-terminal domain of SLPI; Arg58-Ala107) with HNE (human neutrophil elastase). To understand the role of this multiple inhibitory mechanism, the crystal structure of 1/2SLPI with porcine pancreas trypsin was solved and the binding modes of two other complexes compared. The Leu residue surprisingly interacts with the S1 site of trypsin, as with chymotrypsin and elastase. The inhibitory mechanism of 1/2SLPI using the wide primary binding site contacts (from P2' to P5) with various serine proteases is discussed. These inhibitory mechanisms have been acquired in the evolution of the protection system for acute inflammatory diseases.

[Nonspecific IgE potential in nasal discharge for diagnosing allergic rhinitis].

Given that the nasal discharge of subjects with allergic rhinitis contains IgE, we studied nonspecific IgE potential in nasal discharge as a diagnostic marker for allergic rhinitis. We divided 38 adults into 2 groups, one with 22 with Japanese cedar pollinosis and one with 16 with watery rhinorrhea negative to MAST33. The pollinosis group was exposed to Japanese cedar pollen in an environmental exposure unit, and eosinophils in nasal discharge, serum total IgE, pollen-specific IgE, and local IgE in nasal discharge were examined for the 2 groups to determine IgE levels in nasal discharge using the Allerwatch rapid test. In the pollinosis group, nasal discharge IgE correlated significantly with the number of eosinophils. The nasal discharge IgE had higher specificity than the eosinophil examination, whereas nasal discharge eosinophils had higher sensitivity than the IgE examination. We thus found that measuring IgE and eosinophils in nasal discharge is useful for clinically diagnosing allergic rhinitis.

Inhibitory effects of dietary glucosylceramides on squamous cell carcinoma of the head and neck in NOD/SCID mice.

BACKGROUND: Sphingolipids, components of cellular membranes in eukaryotic cells, have roles in the regulation of tumor growth, inflammation, angiogenesis, and immunity. We investigated the effects of dietary glucosylceramides, sphingolipids isolated from rice bran, on tumor growth of human head and neck squamous cell carcinoma. METHODS: The tumor cell line SCCKN cells isolated from well-differentiated human head and neck cancer were subcutaneously inoculated into the right flank of NOD/SCID mice, to establish an SCCKN xenograft model. Rice bran glucosylceramides (300 mg/kg/day) were administered orally to the mice for 14 consecutive days. RESULTS: Dietary glucosylceramides significantly inhibited the growth of the xenograft tumor in comparison with the control group. The TUNEL stain revealed that treatment of mice with glucosylceramides increased the number of apoptotic cells in the implanted tumor tissues and that apoptosis induction was accompanied by the formation of active/cleaved caspase-3. CONCLUSION: These results suggest that dietary glucosylceramides possibly exert anti-tumor activity by inducing apoptosis of head and neck squamous cell carcinoma. Therefore, their potential usefulness in treatment and prevention of human head and neck squamous cell carcinoma warrants further investigation.

Designer µ-oxo-bridged hypervalent iodine(III) organocatalysts for greener oxidations.

The in situ generated µ-oxo-bridged reactive hypervalent iodine(iii) species 1 or their analogues are introduced as more efficient organocatalysts for the catalytic strategy for realizing practical and greener oxidations.

Modification of surgical procedures of type 1 tympanoplasty for non-cholesteatomatous chronic otitis media.

OBJECTIVE: To introduce our modified procedures of type 1 tympanoplasty and to assess their efficacy. METHODS: We modified the surgical procedures of type 1 tympanoplasty and have used these procedures since September 1999. The modified points are enlargement of the facial recess approach, no elevation of the posterior meatal skin and the tympanic annulus, and endoaural repair of tympanic membrane perforation. 51 patients with simple chronic otitis media have undergone this modified type 1 tympanoplasty. Postoperative hearing was evaluated according to the criteria proposed by the Otological Society of Japan. RESULTS: The average follow-up period was 15 months (range 6-35). The hearing result was considered successful when the postoperative hearing level satisfied with at least one of three conditions as follows: (1) air-bone gap <15 dB, (2) hearing gain >15 dB, or (3) hearing level >30 dB. The success rate was 94.1%. The average postoperative air-bone gap, hearing gain and hearing level were 3.9, 10.0 and 29.3 dB, respectively. CONCLUSION: Our modified tympanoplasty is useful to achieve better postoperative hearing results.

Structural analysis of an MK2-inhibitor complex: insight into the regulation of the secondary structure of the Gly-rich loop by TEI-I01800.

Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK2) is a Ser/Thr kinase from the p38 mitogen-activated protein kinase signalling pathway and plays an important role in inflammatory diseases. The crystal structure of the complex of human MK2 (residues 41-364) with the potent MK2 inhibitor TEI-I01800 (pK(i) = 6.9) was determined at 2.9 A resolution. The MK2 structure in the MK2-TEI-I01800 complex is composed of two domains, as observed for other Ser/Thr kinases; however, the Gly-rich loop in the N-terminal domain forms an alpha-helix structure and not a beta-sheet. TEI-I01800 binds to the ATP-binding site as well as near the substrate-binding site of MK2. Both TEI-I01800 molecules have a nonplanar conformation that differs from those of other MK2 inhibitors deposited in the Protein Data Bank. The MK2-TEI-I01800 complex structure is the first active MK2 with an alpha-helical Gly-rich loop and TEI-I01800 regulates the secondary structure of the Gly-rich loop.

Structure of tightly membrane-bound mastoparan-X, a G-protein-activating peptide, determined by solid-state NMR.

The structure of mastoparan-X (MP-X), a G-protein activating peptide from wasp venom, in the state tightly bound to anionic phospholipid bilayers was determined by solid-state NMR spectroscopy. Carbon-13 and nitrogen-15 NMR signals of uniformly labeled MP-X were completely assigned by multidimensional intraresidue C-C, N-CalphaCbeta, and N-Calpha-C', and interresidue Calpha-CalphaCbeta, N-CalphaCbeta, and N-C'-Calpha correlation experiments. The backbone torsion angles were predicted from the chemical shifts of 13C', 13Calpha, 13Cbeta, and 15N signals with the aid of protein NMR database programs. In addition, two 13C-13C and three 13C-15N distances between backbone nuclei were precisely measured by rotational resonance and REDOR experiments, respectively. The backbone structure of MP-X was determined from the 26 dihedral angle restraints and five distances with an average root-mean-square deviation of 0.6 A. Peptide MP-X in the bilayer-bound state formed an amphiphilic alpha-helix for residues Trp3-Leu14 and adopted an extended conformation for Asn2. This membrane-bound conformation is discussed in relation to the peptide's activities to form pores in membranes and to activate G-proteins. This study demonstrates the power of multidimensional solid-state NMR of uniformly isotope-labeled molecules and distance measurements for determining the structures of peptides bound to lipid membranes.

Safe reconstruction of a large cervico-mediastinal tracheal defect with a pectoralis major myocutaneous flap and free costal cartilage grafts.

A large cervico-mediastinal tracheal defect in a 72-year-old man as a result of surgery for thyroid carcinoma with tracheal invasion and mediastinal lymph node metastasis was reconstructed using a pectoralis major myocutaneous flap and free costal cartilage grafts. The tracheal defect (55 mm x 30 mm) was located at the thoracic inlet adjacent to the major mediastinal vessels. Our reconstructive procedure was a two-staged surgery. In the first stage, a pectoralis major myocutaneous flap was transferred to the neck to provide a well-vascularized recipient bed for free costal cartilage grafts and to cover large vessels. Two pieces of free costal cartilage were grafted on the pectoralis major myocutaneous flap, one for the lateral wall reconstruction and the other prefabricated for the anterior wall of the trachea. In the second stage, the re-vascularized cartilage graft for the anterior wall of the trachea with overlying skin was rotated onto the trough of the remaining trachea and the closure of the tracheal defect was completed. We conclude that free cartilage grafts for the reconstruction of a large cervico-mediastinal tracheal defect can be safely used when they are combined with well-vascularized pectoralis major myocutaneous flaps.