Solution structure and ligand-binding site of the carboxy-terminal SH3 domain of GRB2.

BACKGROUND: Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein with three Src homology (SH) domains in the order SH3-SH2-SH3. Both SH3 domains of GRB2 are necessary for interaction with the protein Son of sevenless (Sos), which acts as a Ras activator. Thus, GRB2 mediates signal transduction from growth factor receptors to Ras and is thought to be a key molecule in signal transduction. RESULTS: The three-dimensional structure of the carboxy-terminal SH3 domain of GRB2 (GRB2 C-SH3) was determined by NMR spectroscopy. The SH3 structure consists of six beta-strands arranged in two beta-sheets that are packed together perpendicularly with two additional beta-strands forming the third beta-sheet. GRB2 C-SH3 is very similar to SH3 domains from other proteins. The binding site of the ligand peptide (VPP-PVPPRRR) derived from the Sos protein was mapped on the GRB2 C-SH3 domain indirectly using 1H and 15N chemical shift changes, and directly using several intermolecular nuclear Overhauser effects. CONCLUSIONS: Despite the structural similarity among the known SH3 domains, the sequence alignment and the secondary structure assignments differ. We therefore propose a standard description of the SH3 structures to facilitate comparison of individual SH3 domains, based on their three-dimensional structures. The binding site of the ligand peptide on GRB2 C-SH3 is in good agreement with those found in other SH3 domains.

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.

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.

Kinetic analysis on the substrate specificity of 3-isopropylmalate dehydrogenase.

Substrate specificity of 3-isopropylmalate dehydrogenase is analyzed using a series of synthetic (2R,3S)-3-alkylmalates. Each analog with hydrogen, methyl, ethyl, isopropyl, isobutyl, tert-butyl, and isoamyl group on C-3 functions as a substrate, implying a broad substrate specificity of the enzyme toward alkylmalates. The incremental binding energy of the isopropyl group of 3-isopropylmalate to the enzyme is estimated to be 3.55 kcal/mol, the rather small value supporting the broad specificity. Although the enzyme shows a broad specificity toward the alkylmalates, it does not show activity with isocitrate which has a negatively charged carboxymethyl group instead of the alkyl groups.

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.

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.

Synergistic effects of tamoxifen and cepharanthine for circumventing the multidrug resistance.

We examined the synergistic effects of tamoxifen (TAM) and cepharanthine (CEP) for doxorubicin (DOX) sensitivity using MTT assay. The augmentation of DOX sensitivity by TAM and CEP was significantly correlated with the P-glycoprotein expression. The cytotoxic effect of DOX with TAM and CEP was significantly higher than that of DOX alone, or DOX with TAM, and this synergistic effect was dominant in cell lines with high expression of P-glycoprotein. It was also examined that the intracellular concentration of DOX was increased in combined exposure of TAM and CEP, compared with the exposure of TAM, because TAM and CEP promoted the influx and inhibited the efflux of DOX. Thus, TAM and CEP might be able to circumvent DOX-resistance for treatment in cancer patients.

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.

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.

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.

Evaluation of MTX/5-FU sequential chemotherapy utilized MTT assay for gastrointestinal cancer.

Fresh human gastrointestinal cancer cells are more resistant to anticancer drugs compared to other cancer cells, and the selection of anticancer drugs for cancer chemotherapy is important. In the present study, it is demonstrated that MTX enhanced the chemosensitivity of 5-FU, especially, in the tumor cells with less than 70% inhibition ratio by the MTT assay. It has been reported that MTX/5-FU sequential chemotherapy was one of the effective chemotherapies against gastric cancer and colon cancer, and it is possible to anticipate the efficacy of MTX/5-FU sequential chemotherapy by the MTT assays.

Autologous mixed lymphocyte reaction and postoperative prognosis in patients with gastric carcinoma.

The autologous mixed lymphocyte reaction (AMLR) represents a self recognitive response, which is very important in the immunoregulatory network system. We investigated whether the AMLR activity of patients with gastric carcinoma could reflect the postoperative prognosis to clarify the significance of autoreactivity in anti-tumor immune system in cancer patients. The AMLR activity was suppressed both in the peripheral blood and in the spleen of patients with gastric carcinoma. The patients were divided into two groups; high responder and low responder group. The former consisted of patients whose AMLR activity was extremely suppressed, and the latter of patients whose AMLR activity was mildly suppressed. The survival rate and disease-free survival rate were generally higher in the high responder group than in the low responder group, especially in the spleen. Moreover, none of the patients in the high responder group for the AMLR activity in the spleen died within three years. These results indicated that the AMLR activity could reflect the prognosis of patients who received conventional curative operation. Therefore, it was suggested that the AMLR might be a useful parameter of postoperative prognosis in gastric cancer patients and that autoreactive T cells might play a pivotal role in auto-specific immunological control of tumor growth and metastases.

Intraduodenal osmolality directly enhances insulin secretion in the rat.

To elucidate the direct effect of an intestinal osmolality on insulin release, we investigated the insulin response to intra-duodenal infusion of mannitol in rats. After the anesthesia with intraperitoneal pentobarbital sodium, one milliliter of mannitol solution (10% or 20%) was infused into the duodenum. Portal and femoral blood insulin concentrations significantly increased at 30, 60, and 120 min after intra-duodenal infusion of mannitol, although the blood glucose level did not change. Subcutaneous pre-administration of propranolol (0.4mg/kg) or metoprolol (25mg/kg) completely abolished this phenomenon. These results suggest that intestinal osmolality can directly enhance insulin secretion and that beta 1-adrenergic mechanism is involved in this phenomenon.

In vitro antitumor activity of 4'-O-tetrahydropyranyladriamycin on human gastric cancer cells.

The antitumor activity of 4'-O-tetrahydropyranyladriamycin (THP-ADM) was assessed in established gastric cancer cell lines, including MKN-28, moderately differentiated adenocarcinoma and KATO-III, signet ring cell carcinoma and freshly excised human gastric cancer cells, using the MTT assay. The inhibition rates of THP-ADM were identical to those of adriamycin (ADM) in established gastric cancer cell lines, and the chemosensitivity of MKN-28 was higher than KATO-III. In fresh human gastric cancer cells obtained from 27 patients the inhibition rates of THP-ADM were identical to those of ADM, and there was a significant correlation of inhibition rates ADM and THP-ADM. These results indicate that THP-ADM should be a potent candidate to replace ADM in cancer chemotherapy.

Etoposide enhances the antitumor effects of cisplatin in gastric cancer cells.

We studied the combination effect of cisplatin(CDDP) plus etoposide(VP-16) in an established gastric cancer cell line, KATO-III, and also highly purified fresh human tumor cells obtained from 55 gastric cancer patients, using MTT assay. The synergistic effects of CDDP plus VP-16 were shown by both the fractional product method and median effect plot analysis in KATO-III cells, and by fractional product method in fresh human gastric cancer cells. The combination with CDDP and VP-16 showed the synergistic antitumor effects in not only KATO-III cells, but also fresh human gastric cancer cells. The antitumor effects of CDDP were enhanced by early exposure of VP-16 in KATO-III cells. The combination effects of CDDP and VP-16 were more potent in poorly differentiated gastric cancer, compared with well-differentiated cell types. Thus, it is suggested that the combination of CDDP plus VP-16 is useful in the anticancer chemotherapy of gastric cancer patients.

Hepatic extraction and hepatic action of insulin, glucagon, and epinephrine in bivascularly perfused rat liver.

Using a bivascularly perfused rat liver, we investigated the hepatic extraction and hepatic action on glucose output of insulin, glucagon, and epinephrine. The liver was perfused for 70 min without recirculation via the portal vein and hepatic artery (3.0 ml/(min.g liver) from portal vein and 1.0 ml/(min.g liver) from celiac artery). Low and high concentration of insulin, glucagon, or epinephrine (450 and 4,500 pM, 21.5 and 215 pM, or 410 and 4,100 pM, respectively) was added to the perfusing medium via portal vein (P-liver), via celiac artery (H-liver), or via portal vein and celiac artery (H-P-liver). Hepatic extraction of insulin, glucagon, or epinephrine was not significantly different among H- (56 +/- 9 and 21 +/- 5%, 31 +/- 7 and 24 +/- 6%, or 22 +/- 8 and 17 +/- 5%), P- (50 +/- 10 and 23 +/- 6%, 32 +/- 9 and 23 +/- 7%, or 22 +/- 9 and 18 +/- 6%), and H-P-liver (55 +/- 11 and 24 +/- 6%, 30 +/- 9 and 26 +/- 8%, or 20 +/- 9 and 17 +/- 6%). Glucagon- or epinephrine-induced increase in glucose output was also similar in H- (245 +/- 47 and 498 +/- 82 mumol/30 min, or 112 +/- 21 and 215 +/- 38 mumol/30 min), P- (258 +/- 51 and 512 +/- 95 mumol/30 min, or 128 +/- 20 and 220 +/- 36 mumol/30 min), and H-P-liver (263 +/- 58 and 515 +/- 94 mumol/30 min, or 129 +/- 22 and 225 +/- 39 mumol/30 min).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Temperature and pressure dependence of dielectric properties of ceramic Pb(Fe(1/2)Ta(1/2))O(3).

Dielectric properties such as the real epsilon" and imaginary epsilon" parts of the complex permittivity and the spontaneous polarization P(N) of ceramic Pb(Fe(1/2)Ta(1/2))O(3) with the diffuse phase transition (DPT) were measured under pressures up to 6 kbar. The value of epsilon" shows a broad maximum at a temperature T(m) (mean Curie temperature). As the pressure increases, T(m) of the maximum epsilon" (epsilon"(max)) and the value of epsilon"(max) decrease, and the shape of the maximum in epsilon" around T(m) becomes broader. The pressure coefficient of T(m) is about -3.4 K/kbar. The small heat effect associated with the DPT was observed around T(m). The temperature and pressure dependence of epsilon" and P(s ) is explained in terms of a phenomenological theory using statistical treatments based on a Gaussian distribution of the local Curie temperature. The value of the standard deviation sigma describing the intensity of the DPT is 25 K and increases with increasing pressure.

[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.