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The Phox homology (PX) domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47(phox) PX domain preferably binds phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) and plays a pivotal role in the assembly of phagocyte NADPH oxidase. We describe the PI(3,4)P(2) binding mode of the p47(phox) PX domain as identified by a transferred cross-saturation experiment. The identified PI(3,4)P(2)-binding site, which includes the residues of helices α1 and α1' and the following loop up to the distorted left-handed PP(II) helix, is located at a unique position, as compared with the phosphoinositide-binding sites of all other PX domains characterized thus far. Mutational analyses corroborated the results of the transferred cross-saturation experiments. Moreover, experiments with intact cells demonstrated the importance of this unique binding site for the function of the NADPH oxidase. The low affinity and selectivity of the atypical phosphoinositide-binding site on the p47(phox) PX domain suggest that different types of phosphoinositides sequentially bind to the p47(phox) PX domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase.

Mechanisms that enabled primitive cell membranes to self-reproduce have been discussed based on the physicochemical properties of fatty acids; however, there must be a transition to modern cell membranes composed of phospholipids [Budin I, Szostak JW (2011) Proc Natl Acad Sci USA 108:5249-5254]. Thus, a growth-division mechanism of membranes that does not depend on the chemical nature of amphiphilic molecules must have existed. Here, we show that giant unilamellar vesicles composed of phospholipids can undergo the coupled process of fusion and budding transformation, which mimics cell growth and division. After gaining excess membrane by electrofusion, giant vesicles spontaneously transform into the budded shape only when they contain macromolecules (polymers) inside their aqueous core. This process is a result of the vesicle maximizing the translational entropy of the encapsulated polymers (depletion volume effect). Because the cell is a lipid membrane bag containing highly concentrated biopolymers, this coupling process that is induced by physical and nonspecific interactions may have a general importance in the self-reproduction of the early cellular compartments.

Maraviroc binds to the pocket of extracellular loops of the cell surface CCR5 and prevents R5 HIV-1 from using CCR5 as a coreceptor for entry into CD4-positive cells. To evaluate the contribution of the V3 loop structure in gp120 to maraviroc resistance, we isolated maraviroc-resistant variants from the V3 loop library virus (HIV-1(V3Lib)) containing a set of random combinations of 0-10 polymorphic mutations in vitro. HIV-1(V3Lib) at passage 17 could not be suppressed even at 10 μM (>1400-fold resistance), while HIV-1(JR-FL) at passage 17 revealed an 8-fold resistance to maraviroc. HIV-1(V3Lib-P17) contained T199K and T275M plus 5 mutations in the V3 loop, I304V/F312W/T314A/E317D/I318V. The profile of pseudotyped virus containing I304V/F312W/T314A/E317D/I318V in V3 loop alone revealed a typical noncompetitive resistance, although T199K and/or T275M could not confer noncompetitive resistance. This type of library virus is useful for isolation of escape viruses from effective entry inhibitors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Chemokine receptors play pivotal roles for immune cell recruitment to inflammation sites, in response to chemokine gradients (chemotaxis). The mechanisms of chemokine signaling, especially the initiation of the intracellular signaling cascade, are not well understood. We previously identified a cytoplasmic protein FROUNT, which binds to the C-terminal regions of CCR2 and CCR5 to mediate chemokine signaling. Although large amounts of purified protein are required for detailed biochemical studies and drug screening, no method to produce recombinant FROUNT has been reported. In this study, we developed a method for the production of recombinant human FROUNT. Human FROUNT was successfully expressed in Escherichia coli, as a soluble protein fused to the folding chaperone Trigger Factor, with a cold shock expression system. The purified FROUNT protein displayed CCR2 binding ability without any additional components, as demonstrated by SPR measurements. A gel filtration analysis suggested that FROUNT exists in a homo-oligomeric state. This high-yield method is cost-effective for human FROUNT production. It should be a powerful tool for further biochemical and structural studies to elucidate GPCR regulation and chemokine signaling, and also will contribute to drug development., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The patient was a 54-year-old male with a huge advanced rectal cancer tumor. Abdominal CT showed liver metastasis and local progressive cancer of the rectum measuring 13 × 9 × 7 cm in diameter, which invaded the urinary bladder and sacrum. We established a diagnosis of unresectable rectal cancer and then performed sigmoid colostomy. After 16 courses of FOLFOX4, abdominal CT revealed the liver metastases to have disappeared, and the large-sized advanced rectal cancer had also remarkably decreased in size. Consequently, the patient underwent a resection of the rectum while his bladder was preserved. For 2 years 10 months after surgery, no local recurrence or distant metastasis has been observed, and the patient has received no postoperative chemotherapy. FOLFOX may therefore be a useful preoperative chemotherapy for the patients with unresectable primary rectal cancer.

The hyaluronan (HA) receptor CD44 mediates cell adhesion in leukocyte trafficking and tumor metastasis. Our previous nuclear magnetic resonance (NMR) studies revealed that the CD44 hyaluronan-binding domain (HABD) alters its conformation upon HA binding, from the ordered (O) to the partially disordered (PD) conformation. Here, we demonstrate that the HABD undergoes an equilibrium between the O and PD conformations, in either the presence or absence of HA, which explains the seemingly contradictory X-ray and NMR structures of the HA-bound HABD. An HABD mutant that exclusively adopts the PD conformation displayed a higher HA affinity than the wild-type. Rolling of the cells expressing the mutant CD44 was less efficient than those expressing the wild-type, due to the decreased tether frequency and the slow cellular off rate. Considering that the mutant CD44, devoid of the low-affinity state, exhibited impaired rolling, we conclude that the coexistence of the high- and low-affinity states of the HABD is essential for the CD44-mediated rolling., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A 47-year-old man with a diagnosis of paranasal Wegener's granulomatosis was admitted to our hospital for generalized seizures. He had been treated with long-term predonine therapy after the initial onset of Wegener's granulomatosis. The ictal EEG showed generalized spike and wave complexes, mainly presenting in the bifrontal areas. The postictal EEG revealed periodic localized sharp discharges in the left frontal area. At the initial seizure onset, a midline multilobular mass lesion having a heterogeneous enhancement effect was detected in the lower frontal lobe on gadolinium-enhanced T1-weighted imaging (Gd T1-WI). The anterior skull base and bifrontal lobes were encroached by upward contiguous invasion of the midline mass lesion from the ethmoid sinus on Gd T1-WI. The high signal intensity lesions in the bifrontal lobes on T2- and Gd T1-WI resolved with palliative predonine therapy following methylpredonisolone pulse therapy. Recurrent generalized tonic-clonic status epilepticus was caused by the granulomatous lesion encroaching on the frontal lobe with contiguous invasion from the paranasal Wegener's granulomatosis.

The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 beta-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the "fly-casting" mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.

We report 2 cases of hypoxic ischemic encephalopathy with atypical findings on computed tomographic (CT) and magnetic resonance (MR) imaging for the acute to subacute stage. Case 1: A 78-year-old man with larynx cancer suffered cardiac arrest after suffocation. Cardiopulmonary resuscitation, was performed; the patient then went into a deep coma and also developed severemyoclonus. CT scans on day 0 and day 3 after the arrest revealed no abnormalities MR imaging was performed on day 13 to evaluate cerebral anoxia; however, no abnormal findings were obtained. Since no abnormalities were detected both on CT and MR imaging, we expected that the prognosis would be good; however, the patients did not recover from coma and remains in a persistent vegetative state. Case 2: A 54-year-old man developed cardiac arrest after anaphylactic shock caused by insect bite. After cardiopulmonary resuscitation, the patient lapsed into a deep coma. CT scans performed on day 1 and 3 after the cardiac arrest revealed slight subarachnoid hemorrhage at the surface of the right cerebral cortex along the falx cerebri. MR images obtained on day 10 demonstrated slight hemorrhage at the surface of right cerebral cortex, but no abnormalities in basal ganglia, thalamus, cortex, and white matter. The transient damage of the blood brain barrier caused by hypoxia and ischemia was thought to induce the slight subarachnoid hemorrhage after cardiopulmonary resuscitation. The patient demonstrated early recovery and was almost completely recovered with slight agnosia. MR imaging to rule out hypoxic-ischemic encephalopathy may not have been timed appropriately in both the case, our radiological findings are usual as compare to the findings presented in other similar reports. The variations in the findings of CT and MR imaging in the case of hypoxic-ischemic encephalopathy should be clarified, and the prognosis and management of this condition should be planned on the basis of not only the neuroradiological images but also the neurological signs and symptoms.

During tumor cell invasion, certain extracellular matrix (ECM) components such as hyaluronan (HA) are degraded into small oligosaccharides, which are detected in patients. We previously reported that such HA oligosaccharides induce the proteolytic cleavage of an ECM-binding molecule CD44 from tumor cells and promote tumor cell migration in a CD44-dependent manner. Here, we report that chondroitin sulfate E (CSE), another component of the tumor ECM, strongly enhances CD44 cleavage and tumor cell motility when degraded into oligosaccharides. CSE and its degradation products were detected in pancreatic ductal adenocarcinoma. In CD44-expressing pancreatic tumor cells, degraded forms of CSE but not intact CSE enhanced CD44 cleavage; enzymatic digestion of such low-molecular weight CSE (LMW-CSE) abrogated this enhancement. Among the LMW-CSE preparations examined, 3-kDa CSE most potently induced CD44 cleavage. Nuclear magnetic resonance analysis showed that the 3-kDa-CSE bound to CD44, and that blocking such binding abrogated the CD44 cleavage induction. LMW-CSE also induced prominent filopodia formation and cytoskeletal changes in tumor cells; these effects were also abrogated by blocking the LMW-CSE binding to CD44. Chemically synthesized CSE hexasaccharides also enhanced the CD44 cleavage and tumor cell motility in a CD44-dependent manner. We conclude that the degraded forms of CSE modulate cell adhesion and migration by interacting with tumor-cell CD44, suggesting that the degradation products of tumor-associated ECMs that interact with CD44 play a significant role in CD44-mediated tumor progression.

Collision cancer of the bile duct and the papilla of Vater is an extremely rare entity. This is the first report of a case of bile duct collision cancer. A 75-year-old man presented with jaundice. Computed tomography showed isodensity masses in the middle bile duct and the papilla of Vater. Magnetic resonance cholangiopancreatography showed a tuberous filling defect in the middle bile duct. Gastroduodenal endoscopy showed a tumor with ulceration at the papilla of Vater. The patient was diagnosed with cancers of the middle bile duct and the papilla of Vater, and a pylorus-preserving pancreatoduodenectomy was performed. On pathological examination, the tumor in the middle bile duct showed a well differentiated carcinoma that had spread to the proximal bile duct, whereas the tumor in the papilla of Vater showed a papillo-tubular carcinoma with a marked production of mucin, suggesting an intestinal type of ampullary cancer. These tumors were directly communicated by microscopic findings. Therefore, the immunohistochemical characteristics were analyzed, using several antibodies, to determine whether the origins of the 2 cancers were different or not. As a result, it was concluded that this was a case of collision cancer of the middle bile duct and the papilla of Vater.

Background/aims: It has been thought that intrahepatic stones are brown pigment stones (bilirubin carbonate stones) but we analyzed a chemical compound to reveal that intrahepatic stones have unique components, and studied their pathogenesis., Methodology: A total of 45 gallbladder stones (15 cholesterol stones, 15 black pigment stones, and 15 brown pigment stones) and 15 intrahepatic stones were analyzed about amounts of fatty acids, bile acids and trace elements in the gallstones. Thus we established the characteristic components of the intrahepatic stones and studied their pathogenesis., Results: Concerning the amounts of free fatty acids contained in the gallstones, comparing the 247.2 +/- 116.3 mg/g in the brown pigment stones to the 382.8 +/- 176.3 mg/g in the intrahepatic stones, demonstrates a significant difference between the two groups (p = 0.0191). The ratio of free saturated fatty acids/free unsaturated fatty acids was 1.3 +/- 0.5 in the cholesterol stones, 1.3 +/- 0.6 in the black pigment stones, 5.2 +/- 2.8 in the brown pigment stones and 8.3 +/- 3.5 in the intrahepatic stones (p = 0.0086)., Conclusions: It became clear that the intrahepatic stones contained high levels of free bile acids and that bacterial infection, which deconjugates the glycine and taurine conjugations, is involved in the pathogenesis of gallstones. The fatty acid analysis demonstrated high levels of free saturated fatty acids in the gallstones as well as the involvement of phospholipases, which break down phospholipids in bile, particularly phospholipase A1.

A 82 year-old male having a long history of hypertension was admitted for dysarthria. Neurological examination revealed dysarthria with mild disturbance of left-sided soft palate elevation. No lingual palsy nor facial weakness were noted. No motor weakness in the upper and lower extremities was noted. Diffusion-weighted image and T2 weighted image revealed a small high signal lesion localized in the medial one-third of the left cerebral peduncle. There were bilateral stenotic lesions of the posterior cerebral artery at P1 portion in intracranial magnetic resonance angiography. Pure dysarthria can be caused by disruption of the supranuclear fiber of glossopharyngeal and vagal nerve nucleus in the corticobulbar tract, which can be localized in the medial portion of the cerebral peduncle.

Background/purpose: We aimed to investigate predictors of survival in patients with resectable locally invasive pancreatic cancer., Methods: The patient cohort consisted of 55 patients with locally invasive pancreatic cancer (International Union Against Cancer [UICC] stage III in 36 patients and stage IV in 19) who had undergone resection. The patients were informed about the advantages and the adverse effects of postoperative chemotherapy, and prospectively selected either observation alone or postoperative chemotherapy. The postoperative chemotherapy regimen options were: (1) intraarterial chemotherapy alone, (2) systemic chemotherapy alone, or (3) intraarterial chemotherapy combined with systemic chemotherapy., Results: Overall 1-year and 2-year survival rates after resection were 40.5% and 13.5%, respectively. Median survival time was 10.9 months. Twenty-nine patients (52.7%) received postoperative chemotherapy. On univariate analysis, only postoperative chemotherapy was associated with long-term survival (P < 0.01). In the patients with postoperative chemotherapy, the 1-year survival rate and MST were 61.7% and 16.3 months, compared with 20.1% and 7.9 months in the patients without postoperative chemotherapy. Multivariate analysis also showed that only postoperative chemotherapy was identified as an independent survival factor., Conclusions: It was suggested that postoperative chemotherapy was essential for the improvement of survival in patients with locally invasive pancreatic cancer.

Background/purpose: We aimed to investigate the appropriateness of inserting an intraperitoneal drainage tube after laparoscopic cholecystectomy (LC), based on postoperative pain and clinical courses, in a randomized comparative study., Methods: One hundred and twenty patients who were to have LC were enrolled in this prospective randomized study. An 8-mm Penrose drain was retained below the liver bed for 42 h in each of 60 patients (group A), and no drain was retained in the remaining 60 patients (group B). Patients in each group were hospitalized for 4 days after operation, and the pain reported by the patients, using a visual analogue pain scale (VAS), and the time courses of changes in the highest body temperature, leukocyte count, and C-reactive protein (CRP) were studied comparatively for men and women., Results: Mean VAS scores were significantly greater in group A than in group B at 24 h (P = 0.00004), and 48 h (P = 0.0014) after operation. When sex-stratified changes in mean VAS scores were compared within group A, females had more pain than their male counterparts at 24 h (P = 0.030), but group B showed no sex differences. When the number of patients who used analgesics postoperatively was compared between groups A and B, analgesics were used more frequently in group A. When changes in maximum body temperature were compared, the change was significantly higher in group A than in group B on day 2 after the operation (P = 0.017)., Conclusions: Postoperative pain was intensified by the insertion of a drainage tube after LC. This tendency was stronger in women.

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

Objective: The aim of this study was designed to determine whether the period of drain insertion influences the incidence of postoperative complications., Background Data: The significance of prophylactic drains after pancreatic head resection is still controversial. No report discusses the association of the period of drain insertion and postoperative complications., Methods: A total of 104 consecutive patients who underwent pancreatic head resection were enrolled in this study. To assess the value of prophylactic drains, we prospectively assigned the patients into 2 groups: group I underwent resection from January 2000 to January 2002 (n = 52, drain to be removed on postoperative day 8); group II underwent resection from February 2002 to December 2004 (n = 52, drain to be removed on postoperative day 4). Postoperative complications in the 2 groups were compared., Results: The rate of pancreatic fistula was significantly lower in group II (3.6%) than in group I (23%) (P = 0.0038). The rate of intra-abdominal infections, including intra-abdominal abscess and infected intra-abdominal collections, was significantly reduced in group II (7.7%) compared with group I (38%) (P = 0.0003). Eighteen of 52 (34.6%) patients in group I had an inserted drain beyond 8 days, whereas only 2 of 52 (3.7%) patients in group II had an inserted drain beyond 4 days (P = 0.0002). Cultures of drainage fluid were positive in 16 of 52 (30.8%) patients in group I, and in 2 of 52 (3.7%) patients in group II (P = 0.0002). Intraoperative bleeding (> 1500 mL), operative time (> 420 minutes, and the period of drain insertion were significant risk factors for intra-abdominal infections (P = 0.043, 0.025, 0.0003, respectively). The period of drain insertion was the only independent risk factor for intra-abdominal infections by multivariate analysis (odds ratio, 6.7)., Conclusion: Drain removal on postoperative day 4 was shown to be an independent factor in reducing the incidence of complications with pancreatic head resection, including intra-abdominal infections.

In our study, we investigated whether postoperative chemotherapy improved survival in patients with invasive ductal carcinoma of the pancreas. Between 1987 and 2004, 111 patients underwent pancreatic resection against invasive ductal carcinoma of the pancreas in Wakayama Medical University Hospital. Median survival time (MST) was 19.4 months, 8.6 months, and 7.2 months, in JPS Stage III (UICC Stage IIA and IIB), JPS Stage IVa (UICC Stage IIA and IIB), and JPS Stage IVb (UICC Stage IV), respectively (P < 0.01). The MST of the chemotherapy group was 12 months, and the MST of the non-chemotherapy group was 8.4 months (P < 0.05). Moreover, in JPS Stage IV (UICC Stage IIA, IIB, III, and IV) highly advanced pancreatic cancer, the MST of the chemotherapy group was 10.9 months, and the MST of the group without chemotherapy was 6.6 months (P < 0.01). Since pancreatic cancer is characterized by an aggressive tumor with a high recurrent rate, postoperative chemotherapy is effective for an improvement of survival., (Copyright 2006 Wiley-Liss, Inc.)

Objective: To determine if an antecolic or a retrocolic duodenojejunostomy during pylorus-preserving pancreaticoduodenectomy (PpPD) was associated with the least incidence of delayed gastric emptying (DGE), in a prospective, randomized, controlled trial., Summary Background Data: The pathogenesis of DGE after PpPD has been speculated to be related to factors such as inflammation, ischemia, gastric atony, motilin levels, and type of surgical procedure. Previous retrospective studies have shown a lower incidence of DGE after antecolic duodenojejunostomy. A prospective trial is needed., Methods: Forty patients were enrolled in this trial between May 2002 and April 2004. Just before duodenojejunostomy during PpPD, the patients were randomly assigned to undergo either an antecolic or a retrocolic duodenojejunostomy., Results: DGE occurred in 5% of patients with the antecolic route for duodenojejunostomy versus 50% with the retrocolic route (P = 0.0014). Those with the antecolic route had a significantly shorter duration of postoperative nasogastric tube drainage than did those with the retrocolic route (4.2 days versus 18.9 days, respectively, P = 0.047). By postoperative day 14, all patients with the antecolic route could take solid foods, while only 55% (11 of 20) of the patients with the retrocolic route could take solid foods (P = 0.0007). The length of stay in the hospital was 28 days for the antecolic group versus 48 days for the retrocolic group (P = 0.018)., Conclusions: Antecolic reconstruction for duodenojejunostomy during PpPD decreases postoperative morbidity and length of hospital stay by decreasing DGE. Our data suggest that PpPD with antecolic duodenojejunostomy is a safer operation.

Cyclobutane pyrimidine dimer (CPD) photolyases use light to repair CPDs. For efficient light absorption, CPD photolyases use a second chromophore. We purified Thermus thermophilus CPD photolyase with its second chromophore. UV-visible absorption spectra, reverse-phase HPLC, and NMR analyses of the chromophores revealed that the second chromophore of the enzyme is flavin mononucleotide (FMN). To clarify the role of FMN in the CPD repair reaction, the enzyme without FMN (Enz-FMN(-) and that with a stoichiometric amount of FMN (Enz-FMN(+)) were both successfully obtained. The CPD repair activity of Enz-FMN(+) was higher than that of Enz-FMN(-), and the CPD repair activity ratio of Enz-FMN(+) and Enz-FMN(-) was dependent on the wavelength of light. These results suggest that FMN increases the light absorption efficiency of the enzyme. NMR analyses of Enz-FMN(+) and Enz-FMN(-) revealed that the binding mode of FMN is similar to that of 7,8-didemethyl-8-hydroxy-5-deazariboflavin in Anacystis nidulans CPD photolyase, and thus a direct electron transfer between FMN and CPD is not likely to occur. Based on these results, we concluded that FMN acts as a highly efficient light harvester that gathers light and transfers the energy to FAD.

This study was conducted retrospectively to examine the efficacy of Traverso reconstruction compared with Billroth I reconstruction after pylorus-preserving pancreaticoduodenectomy, in the prevention of several complications. Pylorus-preserving pancreaticoduodenectomy is an aggressive surgery, and insufficiency of the pancreaticoenterostomy plays an important role in the postoperative progression. However, reports examining the correlation between pancreatic fistula and the type of reconstruction after pylorus-preserving pancreaticoduodenectomy have been limited. Sixty-four patients who underwent pylorus-preserving pancreaticoduodenectomy (33 reconstructed by the Traverso technique and 31 reconstructed by the Billroth I technique) were entered into this study to investigate whether the complications were related to the type of reconstruction procedure employed. Insufficiency of the pancreaticojejunostomy, including major leakage and pancreatic fistula, occurred in 18.2% of the reconstructions by Billroth I and 0% of the reconstructions by Traverso (p < 0.05). In addition, jejunal obstruction by recurrent tumor in the remnant pancreas was observed in 3 patients reconstructed by Billroth I, and required palliative bypass surgery. Reconstruction by the Traverso procedure after pylorus-preserving pancreaticoduodenectomy is a safe surgical method and has an advantage for advanced pancreatic cancer, which has high risk of jejunal obstruction by recurrent tumor in the remnant pancreas.

Background/aims: Studies on post-hepatectomy prognoses by infecting viral species have only been rarely reported., Methodology: The patients who had undergone hepatectomy for primary hepatocellular carcinoma (HCC) over the past 10 years at our hospital were divided into three groups based on their underlying causal diseases: hepatitis B (B type), hepatitis C (C type), and non-viral hepatitis including alcoholic hepatitis, and their backgrounds and long-term results after hepatectomy were comparatively investigated., Results: As for tumor factors, the tumor diameter was 3.7+/-2.1 cm in C type patients, which was significantly smaller than the 5.2+/-3.3 cm diameter in B type patients (p=0.01) and 7.9+/-5.6 cm in non-viral patients (p=0.001). However, the frequencies of intrahepatic metastasis, portal vein infiltration, capsule formation and capsule infiltration did not differ significantly among the three groups. Although there was no significant difference observed among the three groups for the recurrence-free survival time, HCC had recurred at as early a stage as within 1 postoperative year in 50% of non-viral patients., Conclusions: The size of a tumor upon hepatectomy and the presence or absence of intrahepatic metastasis were found to be significant factors contributing to the postoperative recurrence of HCC. It is possible that the recurrence-free postoperative survival time may be prolonged by earlier detection of HCC, particularly for the B type and the non-viral type in which the tumor size was already large at the time of surgery.

Cyclobutane pyrimidine dimer (CPD) photolyases, which contain FAD as a cofactor, use light to repair CPDs. We performed structural analyses of the catalytic site of the Thermus thermophilus CPD photolyase-DNA complex, using FAD-induced paramagnetic relaxation enhancement (PRE). The distances between the tryptophan residues and the FAD calculated from the PRE agree well with those observed in the x-ray structure (with an error of <3 A). Subsequently, a single-stranded DNA containing 13C-labeled CPD was prepared, and the FAD-induced PRE of the NMR resonances from the CPD lesion in complex with the CPD photolyase was investigated. The distance between the FAD and the CPD calculated from the PRE is 16 +/- 3 A. The FAD-induced PRE was also observed in the CPD photolyase-double-stranded DNA complex. Based on these results, a model of the CPD photolyase-DNA complex was constructed, and the roles of Arg-201, Lys-240, Trp-247, and Trp-353 in the CPD-repair reaction are discussed.

To improve the metabolic stability of 3, which exhibited both in vitro antitumor activity and in vivo efficacy by both iv and po administration, we designed and synthesized new taxane analogues. Most of the synthetic compounds maintained excellent antitumor activity and were scarcely metabolized by human liver microsomes. And some compounds exhibited potent antitumor effects against B16 melanoma BL6 in vivo by both iv and po administration similarly to 3.

The highest prevalence rate of spinocerebellar ataxia type 6 (SCA6) in the worldwide population is in the Chugoku and Kansai areas of Western Japan, but the reason of this geographic characteristics is unclear. We investigated the predisposing haplotypes and their geographic distribution. Genotyping of five microsatellite markers and three single nucleotide polymorphisms linked to the CACNA1A gene in 150 Japanese SCA6 patients from unrelated 118 families revealed three major haplotypes, carrying a pool of one common haplotype core. A founder chromosome was thought to have historically diverged into at least three types. One of the major haplotypes newly identified showed a strong geographical cluster around the Seto Inland Sea in the Chugoku and Kansai areas of Western Japan, whereas the others were widely distributed throughout Japan. The distribution of predisposing haplotypes contributes to the geographical differences in prevalence of SCA6.

Background: Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant cerebellar ataxia caused by expansion of CAG/CAA trinucleotide repeats in the TATA-binding protein (TBP) gene. Because the number of triplets in patients with SCA17 in previous studies ranged from 43 to 63, the normal number of trinucleotide units has been considered to be 42 or less. However, some healthy subjects in SCA17 pedigrees carry alleles with the same number of expanded repeats as patients with SCA17., Objective: To investigate the minimum number of CAG/CAA repeats in the TBP gene that causes SCA17., Design: We amplified the region of the TBP gene containing the CAG/CAA repeat by means of polymerase chain reaction and performed fragment and sequence analyses., Patients: The subjects included 734 patients with SCA (480 patients with sporadic SCA and 254 patients with familial SCA) without CAG repeat expansions at the SCA1, SCA2, Machado-Joseph disease, SCA6, SCA7, or dentatorubral-pallidolluysian atrophy loci, with 162 healthy subjects, 216 patients with Parkinson disease, and 195 with Alzheimer disease as control subjects., Results: Eight patients with SCA possessed an allele with more than 43 CAG/CAA repeats. Among the non-SCA groups, alleles with 43 to 45 repeats were seen in 3 healthy subjects and 2 with Parkinson disease. In 1 SCA pedigree, a patient with possible SCA17 and her healthy sister had alleles with 45 repeats. A 34-year-old man carrying alleles with 47 and 44 repeats (47/44) had developed progressive cerebellar ataxia and myoclonus at 25 years of age, and he exhibited dementia and pyramidal signs. He was the only affected person in his pedigree, although his father and mother carried alleles with mildly expanded repeats (44/36 and 47/36, respectively). In another pedigree, 1 patient carried a 43-repeat allele, whereas another patient had 2 normal alleles, indicating that the 43-repeat allele may not be pathologic in this family., Conclusions: We estimate that 44 CAG/CAA repeats is the minimum number required to cause SCA17. However, the existence of unaffected subjects with mildly expanded triplets suggests that the TBP gene mutation may not penetrate fully. Homozygosity of alleles with mildly expanded triplet repeats in the TBP gene might contribute to the pathologic phenotype.

Transcription factor IRF-3 is post-translationally activated by Toll-like receptor (TLR) signaling and has critical roles in the regulation of innate immunity. Here we present the X-ray crystal structure of the C-terminal regulatory domain of IRF-3(175-427) (IRF-3 175C) at a resolution of 2.3 A. IRF-3 175C is structurally similar to the Mad homology domain 2 of the Smad family. Structural and functional analyses reveal phosphorylation-induced IRF-3 dimerization, which generates an extensive acidic pocket responsible for binding with p300/CBP. Although TLR and Smad signaling are evolutionarily independent, our results suggest that IRF-3 originates from Smad and acquires its function downstream of TLR.

CD44 is the main cell surface receptor for hyaluronic acid (HA) and contains a functional HA-binding domain (HABD) composed of a Link module with N- and C-terminal extensions. The contact residues of human CD44 HABD for HA have been determined by cross-saturation experiments and mapped on the topology of CD44 HABD, which we elucidated by NMR. The contact residues are distributed in both the consensus fold for the Link module superfamily and the additional structural elements consisting of the flanking regions. Interestingly, the contact residues exhibit small changes in chemical shift upon HA binding. In contrast, the residues with large chemical shift changes are localized in the C-terminal extension and the first alpha-helix and are generally inconsistent with the contact residues. These results suggest that, upon ligand binding, the C-terminal extension and the first alpha-helix undergo significant conformational changes, which may account for the broad ligand specificity of CD44 HABD.

It was shown that a new taxane analogue 3, which exhibited both in vitro antitumor activity and in vivo efficacy by both i.v. and p.o. administration, was prone to be metabolized by human liver microsomes. We identified a major metabolite, M-1, generated by human liver microsomes as 20a, a hydroxylated compound at the pyridine ring of 3. To improve the metabolic stability of 3, we designed and synthesized new taxane analogues based on the structure of M-1, and obtained some compounds that maintained excellent antitumor activity and were scarcely metabolized by human liver microsomes.

We synthesized novel water-soluble and orally active taxane analogues, 7-deoxy-9beta-dihydro-9,10-O-acetal taxanes. Cytotoxicities of the synthetic compounds were greater than those of paclitaxel and docetaxel, especially against resistant cancer cell lines expressing P-glycoprotein. In addition, some compounds showed potent antitumor effects against B16 melanoma BL6 in vivo by both iv and po administration.

The nature of the supramolecular complex between fibrillar collagen and collagen-binding proteins (CBPs) has hindered detailed X-ray and NMR analyses of the ligand-recognition mechanism at atomic resolution because of the lack of appropriate approaches for studying large heterogeneous supramolecular complexes. Recently, we proposed an NMR method, termed transferred cross-saturation (TCS), that enables the rigorous identification of contact residues in a huge protein complex. Here we used TCS to study the supramolecular complex between the A3 domain of von Willebrand factor and fibrillar collagen, which allowed the successful determination of the ligand-binding site of the A3 domain. The binding site of the A3 domain was located at its hydrophobic 'front' surface and was completely different from that of the I domain from the a2 subunit of integrin (alpha2-I domain), which was reported to be the hydrophilic 'top' surface of alpha2-I, although the A3 domain and the alpha2-I domain share a similar fold and possess the identical function of collagen binding.

To investigate structure-activity relationships of the 9,10-acetal-9beta-dihydro taxoids, we modified the 7-hydroxyl groups of the 9,10-acetonide-3'-(4-pyridyl) analogue to deoxy, methoxy, alpha-F, and 7beta,8beta-methano group. As a result of this study, we found that the 7-deoxy analogue was the strongest among these analogues. In addition, we found that the 7-deoxy-3'-(4-pyridyl) and 7-deoxy-3'-(2-pyridyl) analogues showed stronger activity against cell lines expressing P-glycoprotein than the corresponding 3'-phenyl analogue.

A new method for the synthesis of 7-deoxytaxane analogues has been established through hydrogenation of Delta(6,7)-taxane derivatives. Among several catalysts examined, Pd-C was found to be a most effective catalyst for the preparation of target compound.

Using a syngeneic murine model, we investigated the therapeutic efficacy of combined gene therapy using adenoviral vectors expressing murine interleukin-2 (AdmIL-2) and Escherichia coli cytosine deaminase (AdCD). In a subcutaneous tumor model, tumor-bearing mice were treated with an intratumoral injection of adenoviral vectors and received an intraperitoneal administration of 5-fluorocytosine (5-FC). Only the mice treated with AdCD (2 x 10(8) pfu) and an intermediate dose of AdmIL-2 (1 x 10(6) pfu) survived significantly longer than mice treated with AdCD alone (P < 0.01). Moreover, 40% of these treated mice obtained complete remission from tumor-bearing status. The cytotoxicity of splenocytes obtained from the treated mice was related to the survival period. Tumor-specific cytotoxic T lymphocyte assay showed that the cell-mediated cytotoxic response was specific for parental tumor cells. In a hepatic metastasis model, mice treated with an intravenous administration of both AdCD (2 x 10(8) pfu) and an intermediate dose of AdmIL-2 (1 x 10(6) pfu) demonstrated the most significant reduction of metastatic foci and the longest survival following a 5-FC administration. These results suggest that gene therapy combined with AdmIL-2 and AdCD may be a promising strategy for clinical application and, in addition, that translation of combined gene therapy from murine models into the clinical setting will require careful attention to the variables of cytokine expression levels in the design of clinical trials and in the evaluation of treatment efficacy.

In an earlier paper, it was shown that the cross-saturation method enables us to identify the contact residues of large protein complexes in a more rigorous manner than is possible using chemical shift perturbation and hydrogen-deuterium exchange experiments. However, there are limitations within the determination of the contact residues by the cross-saturation method, in that the method is difficult to apply to protein complexes with a molecular mass over 150 kDa and/or with weak binding, since the resonances originating from the complexes should be observed directly in the method. In the present work, to overcome these limitations, we carried out the cross-saturation measurements under conditions of a fast exchange between free and bound states on the NMR time-scale, and determined the contact residues of the complex of the B domain of protein A and intact IgG, which has a molecular mass of 164 kDa and shows weak binding., ((c) 2002 Elsevier Science Ltd.)

To synthesize new highly active taxoids, we designed and synthesized 9 beta-dihydro-9,10-acetal taxoids. In vitro study of these analogues clearly showed them to be more potent than docetaxel.

In the pathological process of Alzheimer's disease (AD), deposition of amyloid beta-peptide (A beta) in the brain parenchyma plays an important role. Neprilysin (NEP), a neutral endopeptidase, degrades A beta, and it is postulated that decreased NEP activity may contribute to the development of AD by promoting the accumulation of A beta. The human NEP gene possesses four dinucleotide repeat polymorphisms, and it is possible that these polymorphisms regulate the NEP expression levels and influence the pathological cascade of AD. Therefore, we investigated the association of these polymorphisms with AD. We performed genotyping of each polymorphism in 201 Japanese sporadic AD patients and 208 Japanese controls. There were no significant differences between the AD and control groups in allele frequencies of each polymorphism. We conclude that these polymorphisms in the NEP gene do not contribute to genetic risk factors for sporadic AD.

One potential target of vaccine therapy for human prostate cancer is the prostate-specific antigen (PSA). One strategy to enhance the immunogenicity of a self-antigen such as PSA is to develop agonist epitopes that are potentially more immunogenic. The studies described here report the design and analysis of an agonist epitope designated PSA-3A ("A" for agonist) of the PSA-3 CTL epitope. Studies demonstrate that when compared with the native PSA-3 epitope, the PSA-3A agonist demonstrates enhanced binding to the MHC class I A2 allele as well as enhanced stability of the peptide-MHC complex. T-cell lines generated with either the PSA-3 or the PSA-3A peptide showed higher levels of lysis of targets pulsed with the PSA-3A peptide than those targets pulsed with the PSA-3 peptide; this was observed when both the concentration of peptide and the ratio of effector to target cells were titrated. T cells stimulated with dendritic cells (DCs) pulsed with PSA-3A peptide produced higher levels of IFN-gamma than did DCs pulsed with PSA-3 peptide; however, no increase in apoptosis was seen in T cells stimulated with the PSA-3A agonist compared with those stimulated with PSA-3. Human T-cell lines generated with the PSA-3A agonist had the ability to lyse human prostate carcinoma cells expressing native PSA in an MHC-restricted manner. Recombinant vaccinia viruses were also constructed that contained the entire PSA transgene with and without the single amino acid change that constitutes the PSA-3A epitope; DCs infected with the recombinant vector containing the agonist amino acid change within the entire PSA gene (designated rV-PSA-3A) were more effective than DCs infected with the rV-PSA vector in enhancing IFN-gamma production by T cells. Finally, the PSA-3A agonist was shown to induce higher levels of T-cell activation, compared with the PSA-3 peptide, in an in vivo model using HLA-A2.1/K(b) transgenic mice. These studies thus demonstrate the potential use of the PSA-3A agonist epitope in both peptide- and vector-mediated immunotherapy protocols for prostate cancer.

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.

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.

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.

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.