Influence of tooth-loss and concomitant masticatory alterations on cholinergic neurons in rats: immunohistochemical and biochemical studies.

The influence of tooth loss on the viability of cholinergic neurons was examined in rats. At 25th postnatal week, rats were divided into the three groups; a control group fed a solid diet, a soft diet group fed a powder diet and a molar crown-less group in which all molar crowns were removed and the powder diet was given. At 15 and 35 weeks post-treatment, the number of choline acetyltransferase (ChAT)-positive neurons in the nucleus of the diagonal band/medial septal nucleus (NDB/MS) was significantly smaller in the molar crown-less group than in the control group (P < 0.01). This was not the case in the pedunculopontine tegmental nucleus or (PPT) or in the trigeminal motor nucleus. Biochemical assay showed no statistically significant differences in choline concentrations in the hippocampus between the control and the molar crown-less group both at 15 and at 35 weeks post-treatment. Nevertheless, acetylcholine (ACh) concentration in the hippocampus of the molar crown-less group was significantly lower than that of the control group at 15 weeks post-treatment (P < 0.05). Taken together, a decrease of oral sensory information may have caused a reduction in the number of ChAT-positive neurons selectively in NDB/MS, which in turn caused a decline of ACh concentrations in the hippocampus.

Difluorinated Danishefsky's diene: a versatile C(4) building block for the fluorinated six-membered rings.

[reaction: see text] A Mg(0)/Me(3)SiCl system was found to be effective for the preparation of difluoro Danishefsky's dienes 2, which involves selective C-F bond cleavage of trifluoromethyl ketones 3. Subsequent hetero Diels-Alder reactions of 2 with aldehydes and imines gave a variety of fluorinated six-membered heterocycles.

Structure and ligand recognition of the PB1 domain: a novel protein module binding to the PC motif.

PB1 domains are novel protein modules capable of binding to target proteins that contain PC motifs. We report here the NMR structure and ligand-binding site of the PB1 domain of the cell polarity establishment protein, Bem1p. In addition, we identify the topology of the PC motif-containing region of Cdc24p by NMR, another cell polarity establishment protein that interacts with Bem1p. The PC motif-containing region is a structural domain offering a scaffold to the PC motif. The chemical shift perturbation experiment and the mutagenesis study show that the PC motif is a major structural element that binds to the PB1 domain. A structural database search reveals close similarity between the Bem1p PB1 domain and the c-Raf1 Ras-binding domain. However, these domains are functionally distinct from each other.

Lack of an association between cystatin C gene polymorphisms in Japanese patients with Alzheimer's disease.

Associations between polymorphisms of the cystatin C gene (CST3) at 5' flanking region and exon 1 in Caucasian patients with late onset AD and exon 1 in a US study of late onset AD have been reported. Clinically diagnosed Japanese patients with AD and Japanese normal control subjects were assessed for the presence of polymorphisms of CST3. The authors could not confirm the previously reported association between CST3 polymorphisms and AD in Japan. Age had no effect on the CST3 genotype.

Enhanced activation of human T cells via avipox vector-mediated hyperexpression of a triad of costimulatory molecules in human dendritic cells.

T-cell activation usually requires at least two signals. The first signal is antigen-specific, and the second signal(s) involves the interaction of a T-cell costimulatory molecule(s) on the antigen-presenting cell (APC) with its ligand on the T cell. Dendritic cells (DCs) are the most potent APCs, attributable, in part, to their expression of several T-cell costimulatory molecules. Human DCs generated in vitro, however, will vary in methods of generation and maturation and in terms of expression of different phenotypic markers-including costimulatory molecules-among different donors. We report here that a recombinant avipox (fowlpox, rF) vector has been constructed that can efficiently express the transgenes for three human T-cell costimulatory molecules (B7-1, ICAM-1, and LFA-3) as a result of individual early avipox promoters driving the expression of each transgene. This triad of costimulatory molecules (designated TRICOM) was selected because each has an individual ligand on T cells and each has been shown previously to prime a unique signaling pathway in T cells. We report here that rF-TRICOM can efficiently infect human DCs of different states of maturity and hyperexpress each of the three costimulatory molecules on the DC surface without affecting other DC phenotypic markers. Infection of influenza or human papilloma virus 9-mer peptide-pulsed DCs from different individuals, or at different stages of maturity with rF-TRICOM, resulted in enhanced activation of T cells from peripheral blood mononuclear cells of autologous donors after 24 h of incubation with DCS: This enhanced activation was analyzed by both titrating the peptide and differing the DC:effector cell ratios. No effect was observed using the control wild-type avipox vector. No increase in apoptosis was observed in T cells hyperstimulated with the TRICOM vector, and no decrease in interleukin-12 production was seen in lipopolysaccharide-stimulated DCs infected with rF-TRICOM. Antibody-blocking experiments demonstrated that enhanced T-cell activation by TRICOM was attributed to each of the three costimulatory molecules. Peptide-pulsed, rF-TRICOM-infected DCs were also shown to be more effective than peptide-pulsed DCs in activating T cells to 9-mer peptides derived from two relatively weak "self" immunogens, i.e., human prostate-specific antigen and human carcinoembryonic antigen. These studies thus demonstrate for the first time that a vector that can simultaneously hyperexpress three costimulatory molecules can be used to efficiently infect human DCs, leading to enhanced peptide-specific T-cell activation. The use of this approach for in vitro studies and clinical applications in immunotherapy is discussed.

Orally active docetaxel analogue: synthesis of 10-deoxy-10-C-morpholinoethyl docetaxel analogues.

To improve cytotoxicity of 10-deoxy-10-C-morpholinoethyl docetaxel analogues against various tumor cell lines including resistant cells expressing P-glycoprotein (P-gp), we modified the 7-hydroxyl group to hydrophobic groups (methoxy, deoxy, 6,7-olefin, alpha-F, 7-beta-8-beta-methano, fluoromethoxy). Among these analogues, the 7-methoxy analogue showed the strongest cytotoxicity. This analogue showed potent activity against B16 melanoma BL6 in vivo by oral administration.

[A case of alcoholic multiple nervous system degeneration].

Chronic ethyl alcohol abuse is associated with different types of neurological involvement. We report a 51-year-old woman with alcoholic encephalopathy, neuropathy and autonomic dysfunction. After the alcohol abuse of about thirty years, gait disturbance, dysphagia and dysarthria progressively worsened. We thought that the disease was caused by poor nutrition due to chronic alcohol abuse and vitamin B1, B12 deficiency. Her neurological symptoms and signs improved after discontinuation of alcohol and nutritional treatment.

Influence of diet and occlusal support on learning memory in rats behavioral and biochemical studies.

In order to verify the relationship between tooth loss and the learning memory in rat, male Wistar rats (25 weeks old) were divided into three separate groups: a control group (fed with a solid diet); a soft diet group (fed with a powder diet containing the same components as the solid one) and a molarless group (all molars were removed at 25 weeks and then fed with a powder diet). To evaluate both learning ability and memory, rats were tested with a one-way step through type of passive avoidance apparatus divided into light and dark chambers at 40-weeks. After the passive avoidance test, determination of acetylcholine (ACh) concentration of the cerebral cortex and hippocampus was performed. There was no significant difference between the molarless group and the control group in the response latency before the acquisition trails (non-stimulated period). At day 4 and 7 after the acquisition trials, the response latency of the molarless group was significantly shorter than that in the control group (p<0.05). The ACh levels of the molarless group in the cerebral cortex and hippocampus were significantly lower than that of the control group (p<0.05). It was apparent that tooth loss had an association with a loss of discriminative learning ability. This study suggested that the decrease of masticatory function caused by tooth loss leads to a decrease of ACh synthesis resulting in a learning memory disorder.

Antitumor effects of interleukin-2 gene-modified fibroblasts in an orthotopic colon cancer model.

We transduced the interleukin-2 (IL-2) gene into murine fibroblasts BALBCL7 or murine colon cancer CT26 using a retroviral vector. BALBCL7 transduced with IL-2 gene secreted 748 pg/ml of IL-2, whereas IL-2 gene-modified CT26 secreted 1,167 pg/ml of IL-2 (48 h incubation, 1x10(6)/ml). Then, we inoculated gene-modified BALBCL7 and/or CT26 cells into BALB/c female mice, and observed the tumor growth. The tumor growth was inhibited in mice inoculated with parental CT26 plus IL-2 gene-modified BALBCL7, compared with that in mice given parental CT26 alone (P<0.01). Moreover, we investigated the cytotoxic activity of spleen cells derived from mice treated with gene-modified cells, and performed phenotypic analysis of the effector cells. The killer cells derived from mice inoculated with IL-2 gene-modified BALBCL7 plus parental CT26 showed higher cytotoxic activity than those from mice inoculated with CT26 alone. The cytotoxic activity was almost completely blocked by anti-CD8 antibody (Ab), and partially blocked by anti-asialo GM1 Ab. Next, we inoculated CT26 tumor tissue into murine cecum orthotopically, and treated the animals with gene-modified BALBCL7 plus parental CT26. The tumor size in the cecum was significantly decreased, compared with parental CT26 alone (P<0.01).

P-glycoprotein-expressing tumor cells are resistant to anticancer drugs in human gastrointestinal cancer.

The resistance to doxorubicin (DOX) by some tumor cells is mainly due to the effect of P-glycoprotein encoded by the multidrug resistance-1 (mdr1) gene. We tried to prove the correlations between P-glycoprotein expression and the sensitivity for anticancer drugs including DOX and other cytotoxic drugs that are currently used for gastrointestinal cancer patients. We quantified the P-glycoprotein expression by flow cytometry techniques, and the sensitivity for anticancer drugs using a tetrazolium salt, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), assay in highly purified fresh human tumor cells obtained from 25 cancer patients. The inhibition rates were the lowest in DOX and mitomycin C (MMC), compared with other drugs. The most significant correlation between DOX and MMC was seen in the inhibition rates. A significant correlation was also seen between the inhibition rates for DOX and P-glycoprotein expression, whereas only a slight correlation between the sensitivity for MMC and P-glycoprotein expression was observed. We should therefore pay close attention to the effect of P-glycoprotein when treating cancer patients, especially if both the inhibition rates of DOX and MMC are low based on the findings of an MTT assay.

Tetratricopeptide repeat (TPR) motifs of p67(phox) participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase.

The small GTPase Rac functions as a molecular switch in several important cellular events including cytoskeletal reorganization and activation of the phagocyte NADPH oxidase, the latter of which leads to production of superoxide, a precursor of microbicidal oxidants. During formation of the active oxidase complex at the membrane, the GTP-bound Rac appears to interact with the N-terminal region of p67(phox), another indispensable activator that translocates from the cytosol upon phagocyte stimulation. Here we show that the p67(phox) N terminus lacks the CRIB motif, a well known Rac target, but contains four tetratricopeptide repeat (TPR) motifs with highly alpha-helical structure. Disruption of any of the N-terminal three TPRs, but the last one, results in defective interaction with Rac, while all the four are required for the NADPH oxidase activation. We also find that Arg-102 in the third repeat is likely involved in binding to Rac via an ionic interaction, and that replacement of this residue with Glu completely abrogates the capability of activating the oxidase both in vivo and in vitro. Thus the TPR motifs of p67(phox) are packed to function as a Rac target, thereby playing a crucial role in the active oxidase complex formation.

Solution structure of the SH2 domain of Grb2 complexed with the Shc-derived phosphotyrosine-containing peptide.

The solution structure of growth factor receptor-bound protein 2 (Grb2) SH2 complexed with a Shc-derived phosphotyrosine (pTyr)-containing peptide was determined by nuclear magnetic resonance (NMR) spectroscopy. The pTyr binding site of Grb2 SH2 was similar to those of other SH2 domains. In contrast, the amino acid residues C-terminal to pTyr did not form an extended structure because of steric hindrance caused by a bulky side-chain of Trp121 (EF1). As a result, the peptide formed a turn-structure on the surface of Grb2 SH2. The asparagine residue at the pTyr+2 position of the Shc-peptide interacted with the main-chain carbonyl groups of Lys109 and Leu120. The present solution structure was similar to the crystal structure reported for Grb2 SH2 complexed with a BCR-Abl-derived phosphotyrosine-containing peptide. Finally, the structure of Grb2 SH2 domain was compared with those of the complexes of Src and phospholipase C-gamma1 with their cognate peptides, showing that the specific conformation of the peptide was required for binding to the SH2 domains.

Solution structure of the SH2 domain of Grb2/Ash complexed with EGF receptor-derived phosphotyrosine-containing peptide.

1H, 13C, and 15N NMR resonances of the SH2 domain of Grb2/Ash in both the free form and the form complexed with a phosphotyrosine-containing peptide derived from the EGF receptor were assigned by analysis of multi-dimensional, double- and triple-resonance NMR experiments. From the chemical shift changes of individual residues upon peptide binding, the binding site for the peptide was mapped on the structure of Grb2/Ash SH2. The peptide was not recognized by the groove formed by the BG and EF loops, suggesting that the EGFR peptide does not bind to Grb2/Ash SH2 in an extended conformation. This was supported by analysis of the binding affinity of mutants where residues on the BG and EF loops were changed to alanine. The present results are consistent with the recently reported structures of Grb2/Ash SH2 complexed with BCR-Abl and Shc-derived phosphotyrosine containing peptides, where the peptide forms a turn conformation. This shows that the specific conformation of the phosphotyrosine-containing sequence is required for the SH2 binding responsible for downstream signaling.

Choledochoduodenal fistula at the anterior wall of the duodenal bulb: a rare complication of duodenal ulcer.

A 38 year-old man was admitted to our hospital with the chief complaint of epigastralgia. His laboratory data revealed leukocytosis and increased serum amylase, and abdominal ultrasonography revealed diffuse swelling of the pancreas. Thus, he was diagnosed as having acute pancreatitis. Moreover, abdominal computed tomography showed pneumobilia in the gallbladder and the common bile duct. Gastroduodenal fiberscopy demonstrated peptic ulcer scars around a foramen with smooth margins at the anterior wall of the duodenal bulb. The bile juice flowed from the bottom of the foramen. Endoscopic retrograde cholangiopancreatography revealed the fistula between the common bile duct and the anterior wall of the duodenal bulb, but not the posterior wall. However, there was no pancreatico-biliary maljunction and no stones in the gallbladder or bile duct. This is a rare case of choledochoduodenal fistula at the anterior wall of the duodenal bulb caused by duodenal peptic ulcer disease.

P-glycoprotein expression and chemosensitivity in highly purified fresh human gastrointestinal cancer cells.

BACKGROUND/AIMS: Colorectal cancer is one of the tumors most refractory to treatment by chemotherapy. One of the major problems associated with cancer chemotherapy is drug-resistance of tumor cells, and resistance to doxorubicin (DOX) is mainly due to the effect of P-glycoprotein. We have tried to prove the correlation between P-glycoprotein expression and DOX-sensitivity in highly purified fresh human colorectal cancer and, moreover, to prove the differentiation of P-glycoprotein expression between the different kinds of cancers, including gastric cancer. METHODOLOGY: The present study was designed to quantify P-glycoprotein expression by flow cytometry, and DOX-sensitivity by MTT assay in highly purified fresh human tumor cells obtained from 29 cancer patients including 13 colorectal cancers and 16 gastric cancers. RESULTS: DOX-sensitivity decreased in proportion to P-glycoprotein expression in colorectal cancer. P-glycoprotein expression in colorectal cancer was higher than that in gastric cancer. Particularly, P-glycoprotein expression in colorectal cancer in the DOX low-sensitivity group was higher than in the DOX high-sensitivity group. CONCLUSIONS: The chemotherapeutic management of patients with colorectal cancer might be more effective if we can circumvent the effect of P-glycoprotein.

Establishment and analysis of biological characteristics of a human duodenal carcinoma cell line, WDC-1.

BACKGROUND: Duodenal carcinoma is very rare and its culture cell lines have rarely been established. METHODS: Tumor cells separated from a surgically resected primary tumor of duodenal carcinoma were put into culture. The patient was an 81-year-old female and had metastatic lymph nodes. We investigated the biological characteristics of the culture cells including in vitro cell kinetics, karyotype, expression of tumor markers and integrins and tumorigenicity and histology in nude mice. RESULTS: A new cell line, designated WDC-1, was established. This duodenal carcinoma cell line proliferated in a monolayered sheet with a doubling time of 50 h. The histological findings of the xenograft in nude mice were similar to those of the primary tumor. WDC-1 cells produced carcinoembryonic antigen and expressed 1 integrin and very late antigen (VLA)-4d in vitro. CONCLUSIONS: A duodenal carcinoma cell line was established, which is rare and may contribute to progress in understanding the biological features of duodenal cancer.

Synthesis and evaluation of water-soluble non-prodrug analogs of docetaxel bearing sec-aminoethyl group at the C-10 position.

To develop non-prodrugs of taxoids with satisfactory stability in vivo, high water-solubility, and potent antitumor activity, we prepared several 10-O-sec-aminoethyl docetaxel analogs (3) and evaluated their cytotoxicity against mouse leukemia and human tumor cell lines, microtubule disassembly-inhibitory activity, and water-solubility. These analogs were synthesized from the 10-O-allyl baccatin derivatives (5a-c) using the beta-lactam synthon method. Among these analogs, the 10-O-(2-morpholinoethyl) (18, 21) and 10-O-(2-thiomorpholinoethyl) (19, 24) analogs exhibited cytotoxicity comparable or superior to that of docetaxel (2). In addition, the methanesulfonic acid salt (18a) had a high water-solubility.

Synthesis and antitumor activity of ring A- and F-modified hexacyclic camptothecin analogues.

Nineteen ring A- and F-modified hexacyclic analogues of camptothecin were synthesized by Friedländer condensation of appropriately substituted bicyclic amino ketones with tricyclic ketone and were evaluated for cytotoxicity and topoisomerase I inhibitory activity. Seventeen of the compounds showed cytotoxic effects comparable or superior to those of 7-ethyl-10-hydroxycamptothecin (SN-38) against mouse leukemia P388 and human tumor cell lines HOC-21 and QG-56. Introduction of a compact and inductively electron-withdrawing substituent such as a hydroxy, methoxy, chloro, or fluoro group into position 5 of ring A of the hexacyclic compound remarkably increased the antitumor activity. The potency of topoisomerase I inhibition of these compounds showed good correlation with their cytotoxicity. Among them, the 4-methyl-5-fluoro hexacyclic compound was the most potent of all and was 10 times as active as SN-38 in in vitro antitumor activity.

Intraoperative radiation therapy for a patient with bulky disease of mesenteric non-Hodgkin's lymphoma.

A long survival is rarely observed in patients demonstrating recurrent malignant lymphoma with bulky disease because of the appearance of chemoresistant tumor cells after extensive chemotherapy, and moreover the presence of bulky disease has also been consistently associated with a poorer response rate and a shortened survival, due to the fact that tumor size is the most significant factor for the treatment of non-Hodgkin's lymphoma. We herein describe a case of a 53-year-old woman presenting with the chief complaint of abdominal fullness, who underwent intraoperative radiation therapy (IORT) for recurrent bulky non-Hodgkin's lymphoma in the mesenterium. The patient has had no evidence of tumor recurrence, based on the findings of regular abdominal computed tomographic scans, 60 months after initial chemotherapy and 28 months after IORT.

Synthesis and cytotoxic activity of novel 10-alkylated docetaxel analogs.

An alkylation method of docetaxel at the C-10 position has been established by a radical coupling reaction using a 10-xanthate derivative of 7-O-TES-10-deacetylbaccatin III and appropriate alkenes. In addition the cytotoxic activity of 10-alkylated docetaxel analogs was evaluated. Among these analogs, a derivative having a methoxycarbonyl group at the end of the alkyl moiety exhibited more potent cytotoxic activity than docetaxel.