Effects of extracellular DNA and DNA-binding protein on the development of a Streptococcus intermedius biofilm.

AIMS: The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone-like DNA-binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity. METHODS AND RESULTS: Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7-hydoxyl-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) and anti-HLP antibody without fixation, respectively. DNase I treatment (200 U ml(-1)) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 µg ml(-1)) purified from Strep. intermedius, other Gram-positive bacteria, Gram-negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild-type, HLP-downregulated strain and control strains. In contrast, the addition of eDNA (>1 µg ml(-1)) decreased the biofilm mass of all Strep. intermedius strains. CONCLUSIONS: These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity. SIGNIFICANCE AND IMPACT OF THE STUDY: eDNA- and HLP-targeting strategies may be applicable to novel treatments for bacterial biofilm-related infectious diseases.

Bactericidal activity and oral pathogen inactivation by electromagnetic wave irradiation.

AIMS: The aim of this work was to clarify the effects of electromagnetic wave irradiation (EMWI) on oral bacterial pathogens. METHODS AND RESULTS: A Gram-negative (Porphyromonas gingivalis) or Gram-positive (Streptococcus mutans, S. intermedius, Enterococcus faecalis) bacterial suspension was irradiated by EMW apparatus (500-1000 kHz, 5-15 times, 1 s time(-1) ). Quantification of survival bacteria by CFU counting revealed that EMWI exhibited marked bactericidal activity against all tested bacteria and bactericidal activity at 500 kHz increased in an irradiation number-dependent manner. After EMWI at 500 kHz, scanning electron microscopic observations showed that the chain of S. mutans cells was shortened after 5 irradiations and the outlines of bacterial cells (S. mutans and P. gingivalis) were unclear after 5-10 irradiations. EMWI inhibited the inductive effect of S. mutans on pro-inflammatory cytokine production in human monocytes and this inhibitory effect was comparable with that of heat-killed bacteria. Furthermore, using an enzyme activity assay, EMWI partially inactivated the activities of gingipains from P. gingivalis. CONCLUSIONS: These findings demonstrated that EMWI has inactivation and bactericidal activities against single microbial species among four kinds of oral pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Electromagnetic wave irradiation may be applicable for medical disinfection and sterilization, such as refractory periapical periodontitis.

Roles of TLR2, TLR4, NOD2, and NOD1 in pulp fibroblasts.

Pulp fibroblasts express various pro-inflammatory mediators leading to marked infiltration of inflammatory cells in the progression of pulpitis. We hypothesized that pulp fibroblasts play roles in the recognition of invaded caries-related bacteria and the subsequent innate immune responses. We found clear expressions of TLR2, NOD1, and NOD2 and a faint expression of TLR4 in human dental pulp fibroblasts (HDPF) by RT-PCR and flow cytometry. We also observed that various pro-inflammatory mediators, including cytokines, chemokines, adhesion molecules, prostaglandin E(2) and its key enzyme COX-2, not iNOS or caspase-1, were markedly up-regulated by stimulation with these TLR and NOD agonists. More over, the NOD2 agonist acted synergistically with the TLR2, not the TLR4, agonist to stimulate the production of pro-inflammatory mediators in HDPF. These findings indicate that TLR2, TLR4, NOD2, and NOD1 in HDPF are functional receptors, and NOD2 is a modulator of signals transmitted through TLR2 in pulpal immune responses, leading to progressive pulpitis.

Caries-related bacteria and cytokines induce CXCL10 in dental pulp.

UNLABELLED: Marked infiltration of inflammatory cells, such as activated T-cells, is observed in the progression of pulpitis; however, little is known about the mechanism of their recruitment into pulpal lesions. It has been recently demonstrated that CXC chemokine ligand 10 (CXCL10) chemoattracts CXC chemokine receptor 3 (CXCR3)-positive activated T-cells. We therefore examined whether CXCL10 is involved in the pathogenesis of pulpitis. CXCL10 mRNA expression levels in clinically inflamed dental pulp were higher than those in healthy dental pulp. Immunostaining results revealed that CXCL10 was detected in macrophages, endothelial cells, and fibroblasts in inflamed dental pulp, and that CXCR3 expression was observed mainly on T-cells. Moreover, cultured dental pulp fibroblasts produced CXCL10 after stimulation with live caries-related bacteria, peptidoglycans, and pro-inflammatory cytokines. In contrast, heat-killed bacteria did not induce CXCL10 secretion. These findings suggest that CXCL10-CXCR3 may play an important role in the pulpal immune response to caries-related bacterial invasion. ABBREVIATIONS: CXCL10, CXC chemokine ligand 10; CXCR3, CXC chemokine receptor 3; IFN, interferon; FBS, fetal bovine serum; LTA, lipoteichoic acid; PGN, peptidoglycan; IL, interleukin; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; CCL, C-C chemokine ligand; TLR, Toll-like receptor; NOD, nucleotide oligomerization domain; HDPF, human dental pulp fibroblasts.

A phase I study of S-1 combined with weekly cisplatin for metastatic gastric cancer in an outpatient setting.

A dose-escalation study was conducted for patients with metastatic gastric cancer to determine the recommended dose of weekly intravenous (i.v.) cisplatin combined with a fixed dose of a new oral dihydropyrimidine dehydrogenase-inhibitory fluoropyrimidine, S-1, on an outpatient basis. Secondary endpoints were to define the toxicity profile and to determine tumour responses. S-1 was fixed at a dose of 70 mg/m(2)/day and was administered for 2 weeks followed by a 1-week rest. Three dose levels of cisplatin (10, 15 and 20 mg/m(2)) were studied. Cisplatin was infused over 30 min on days 1 and 8. 20 patients were enrolled. No dose-limiting toxicities (DLTs) were recorded during the administration of cisplatin up to 20 mg/m(2), except for grade 3 diarrhoea and stomatitis in one patient at dose level 3. No grade 4 adverse events occurred. However, grade 2 gastrointestinal adverse reactions, such as nausea and anorexia, were seen in 7 of 13 patients at dose level 3 within the first two treatment cycles. This was determined to be the maximum acceptable level that would not negate the advantages observed with use of an oral drug such as S-1. An objective tumour response was seen at all dose levels, and the overall response rate in the 18 patients evaluated was 61%. A higher response rate of 78% was observed in 9 patients who had received no prior chemotherapy. Oral S-1 with weekly cisplatin is a feasible and promising combination regimen that is appropriate for an outpatient setting. A randomised phase II study comparing this combination with S-1 alone in chemo-nai;ve patients is warranted.

Ultrasonographic assessment of coronary flow reserve and abdominal fat in obesity.

Recent technological advances in transthoracic Doppler echocardiography (TTDE) have provided noninvasive measurement of coronary flow velocity reserve (CFVR). We aimed to quantitate a correlation between endothelial dysfunction and fat distribution. In 36 patients with obesity, 16 with noninsulin-dependent diabetes mellitus (DM) and 12 healthy volunteers, coronary flow velocity was measured at the distal site of the left anterior descending branch. CFVR was defined as the ratio of hyperemic (IV infusion of 0.15 mg/kg/min adenosine) to basal peak diastolic flow velocity. Abdominal wall fat index (AWFI) was estimated by ultrasonography. Insulin resistance was quantified by the euglycemic hyperinsulinemic clump method. AWFI was significantly related to CFVR (r = -0.46, p = 0.011) and insulin resistance (r = -0.71, p < 0.0001). CFVR could be noninvasively evaluated using TTDE. Coronary endothelial dysfunction indicated as CFVR, body fat distribution and insulin resistance was quantitatively correlated in obesity.

[A case of renal cell carcinoma producing alpha-fetoprotein].

Renal cell carcinoma (RCC) producing alpha-fetoprotein (AFP) is a rare condition with only 11 cases reported in Japan to our knowledge. A 69-year-old man was admitted to our hospital for further examination of an incidental right renal tumor. Laboratory tests showed markedly increased serum level of AFP whereas both HBs antigen and anti-HCV antibody were negative. Computed tomography and magnetic resonance imaging imagings showed a right renal tumor but no tumor in liver, testis or lymph node. We performed right radical nephrectomy. Serum level of AFP declined within the normal range 7 weeks after nephrectomy according to its half-life curve. The tumor specimen was composed mainly of granular cells. Immunohistochemical examination of the tumor cells proved the presence of AFP in the cytoplasm. The possibility of AFP as a tumor marker of renal cell carcinoma in this case was presented.

Strain variation in renal carcinogenesis by N-ethyl-N-hydroxyethylnitrosamine in F1 (Wistar-Fischer) rats.

The present study was conducted to compare the incidences of renal tumors in Wistar (W), Fischer (F) and F1 rats (WF: female Wistar rats x male Fischer rats; FW: female Fischer rats x male Wistar rats) induced by N-ethyl-N-hydroxyethylnitrosamine (EHEN). Levels of 8-OHdG in renal DNA were also investigated in Wistar and Fischer rats. After 2000 ppm of EHEN was administered orally for 2 weeks, the animals were fed basal diet until week 32. Wistar males and females demonstrated significantly higher sensitivity regarding induction of renal lesions, while both WF and FW rats had similar incidences, generally intermediate between those for the two parent strains. The formation of 8-OHdG was maximal 60-180 min after an intraperitoneal dose of 750 mg/kg to Wistar and Fischer rats, which correlates with the increase tending to the incidence of renal tumors in male and female Wistar and Fischer rats. The results suggest that EHEN induction of renal tumors is related to oxygen radical damage and that the genes in the Wistar strain responsible for the sensitivity are not inherited in a sex-dependent fashion, despite the male being more susceptible.

Evaluation of aberrant bile ducts before laparoscopic cholecystectomy: helical CT cholangiography versus MR cholangiography.

OBJECTIVE: The purpose of our study was to compare the accuracy of helical CT cholangiography and that of MR cholangiography in the diagnosis of aberrant bile ducts or cystic ducts before laparoscopic cholecystectomy. SUBJECTS AND METHODS: A total of 120 consecutive patients, including 114 patients with cholecystolithiasis and six with gallbladder polyps, were treated using laparoscopic cholecystectomy between November 1996 and August 1998. Eighteen (15%) of the 120 patients were suspected of having aberrant bile ducts or cystic ducts on helical CT cholangiography, and 16 of these 18 patients were subsequently examined on MR cholangiography. For the 16 patients who underwent both imaging examinations, findings from helical CT cholangiography and MR cholangiography were compared with intraoperative cholangiography. RESULTS: Aberrant bile ducts in 13 patients and aberrant cystic ducts in three patients were divided into six types on the basis of the results of intraoperative cholangiography. Although these types were clearly identified using helical CT cholangiography in all 16 patients, the anatomic variants were not correctly identified in seven (44%) of the 16 patients with MR cholangiography. False-negative findings were mainly a result of the insertion sites of the cystic ducts or aberrant bile ducts being obscured by aberrant bile ducts or duodenum. Two (2%) of the 120 patients developed mild adverse reactions to the contrast material, but neither required treatment. CONCLUSION: Helical CT cholangiography clearly showed aberrant bile ducts and cystic ducts, but visualization of these structures on MR cholangiography was unsatisfactory because of overlapping duodenum and hepatic ducts.

[Seven cases of brain metastasis from papillary thyroid carcinoma].

Brain metastases from differentiated thyroid carcinoma are extremely rare and carry a poor prognosis. We describe here clinical details of 7 cases of brain metastases from papillary thyroid carcinoma. Of 153 patients with metastases from differentiated thyroid carcinoma (papillary in 123, follicular in 30) treated at our institution between 1981 and 1999, 7 patients (4.6%) had brain metastases. Histologically, the primary tumor was papillary carcinoma in all 7 cases. Four were males and 3 were females. The median age at first diagnosis of distant metastases was 63 yr (range, 47-76 yr). Of these patients, one had brain metastases only and six had metastases to the lungs as well. Five of these patients were treated with 131I. Three of these 5 patients had marked uptake in the metastases (131I positive) on post-therapy 131I scans and another 2 patients had no significant activity (131I negative) in both pulmonary and brain metastatic lesions. One of 3 patients with 131I positive lesions had intense activity in the brain tumor, but no uptake in multiple pulmonary metastatic tumors. In a patient with 131I positive brain metastases, the tumors progressed rapidly after 131I therapy. In another one patient, acute hemorrhage of the tumor occurred four days after 131I therapy, requiring surgical removal. Loner case of 131I negative 2 patients was treated with radiosurgery (gamma-knife) and complete reduction in tumor volume was observed. On the other hand, one of 2 patients receiving no 131I therapy had radiosurgery (x-knife) and remaining one received conventional external radiation and chemotherapy for small solitary brain and pulmonary metastatic tumors. These therapeutic interventions were useful in both cases. The mean length of survival after the development of brain metastases in the five patients who died of the disease was 30 months. One patient treated with x-knife has been alive at 21 months and another one who has 131I uptake in the brain tumor without uptake in lung lesions has been alive 15 months after diagnosis of brain metastasis. These results indicate that it is important to detect brain metastasis by imaging techniques and Tg measurements and give treatment as early as possible since the brain is the third most common distant metastatic site and the prognosis is poor.

PAPIN. A novel multiple PSD-95/Dlg-A/ZO-1 protein interacting with neural plakophilin-related armadillo repeat protein/delta-catenin and p0071.

A neural plakophilin-related armadillo repeat protein (NPRAP)/delta-catenin interacts with one of Alzheimer disease-related gene products, presenilin 1. We have previously reported the interaction of NPRAP/delta-catenin with synaptic scaffolding molecule, which is involved in the assembly of synaptic components. NPRAP/delta-catenin also interacts with E-cadherin and beta-catenin and is implicated in the organization of cell-cell junctions. p0071, a ubiquitous isoform of NPRAP/delta-catenin, is localized at desmosomes in HeLa and A431 cells and at adherens junctions in Madin-Darby bovine kidney cells. We have identified here a novel protein interacting with NPRAP/delta-catenin and p0071 and named this protein plakophilin-related armadillo repeat protein-interacting PSD-95/Dlg-A/ZO-1 (PDZ) protein (PAPIN). PAPIN has six PDZ domains and binds to NPRAP/delta-catenin and p0071 via the second PDZ domain. PAPIN and p0071 are ubiquitously expressed in various tissues and are localized at cell-cell junctions in normal rat kidney cells and bronchial epithelial cells. PAPIN may be a scaffolding protein connecting components of epithelial junctions with p0071.

Kinetic and theoretical studies on the mechanism of alkaline hydrolysis of DNA.

The reaction mechanism of alkaline hydrolysis of DNA has been investigated by kinetic analysis and density-functional-theory calculation. The rates of hydrolysis of thymidine 3'-monophosphate esters (including thymidylyl(3'-5')thymidine (Tp-OT)) monotonically decrease as the leaving groups get poorer. According to the theoretical calculation in which the solvent effects are incorporated, no intermediate is formed in the course of the reaction. In the alkaline hydrolysis of the activated Tp-OT analogues having good leaving groups, the 3',5'-cyclic monophosphate of thymidine is concurrently formed through the intramolecular attack by the 5'-alkoxide ion. In the hydrolysis of the native dinucleotide, however, this side reaction does not occur, since the transition state leading to the departure of its poor leaving group cannot be formed due to conformational restraint. These arguments are supported by the theoretical analysis on the hydrolysis of both dimethyl phosphate and its O(bridging)-->S substituted analogue.

The cervical aortic arch with aneurysm formation.

An asymptomatic 59-year-old man was admitted with an initial suspicion of mediastinal tumor. He was diagnosed as having a left-sided cervical aortic arch (Haughton type D) with arch aneurysm by using contrast-enhanced CT and angiography. The arch aneurysm was surgically removed. This is the 20th reported case of cervical aortic arch with aneurysm formation.

Insulin resistance and classic risk factors in type 2 diabetic patients with different subtypes of ischemic stroke.

OBJECTIVE: In addition to classic risk factors (e.g., hypertension), insulin resistance is an important risk factor for the development of atherosclerosis. To investigate the risk factors for ischemic stroke in type 2 diabetes, we measured insulin sensitivity and several risk factors in 94 Japanese type 2 diabetic patients with different types of stroke. RESEARCH DESIGN AND METHODS: Stroke was classified by magnetic resonance imaging (MRI) and magnetic resonance (MR) angiography into the following subtypes: 1) patients with normal MRI and MR angiography (NOR; n = 30), 2) patients with lacunar infarction (LAC; n = 28), 3) patients with atherothrombotic infarction (ATI; n = 22), and 4) patients with large-artery atherosclerosis (LAA; n = 14). Insulin sensitivity was assessed by the K index of the insulin tolerance test (KITT). RESULTS: Patients with LAC, ATI, and LAA were significantly older and were more likely to be hypertensive than patients with NOR. Significantly higher insulin resistance was observed in patients with LAC, ATI, and LAA than in patients with NOR (KITT 2.21 +/- 0.17, 2.10 +/- 0.17, 2.19 +/- 0.25, and 3.25 +/- 0.21% per min, respectively, P < 0.001). Adjustment for age, sex, BMI, and duration of diabetes did not influence this result. Multiple logistical regression analysis showed that insulin resistance was an independent risk factor for all subtypes of ischemic stroke in type 2 diabetic patients. The same analysis showed that a high pulse pressure was a risk factor for LAC, postprandial C-peptide (hyperinsulinemia) was a risk factor for ATI, and longstanding hyperglycemia was a risk factor for LAA.

Isolation and characterization of mammalian homologues of Caenorhabditis elegans lin-7: localization at cell-cell junctions.

In Caenorhabditis elegans, the vulval induction is mediated by the let-23 receptor tyrosine kinase (RTK)/ Ras signaling pathway. The precise localization of the let-23 RTK at the epithelial junctions is essential for the vulval induction, and requires three genes including lin-2, -7, and -10. The mammalian homologue of lin-2 has been identified as a protein interacting with a neuronal adhesion molecule, neurexin, and named CASK. CASK has recently been reported to interact with syndecans and an actin-binding protein, band 4.1, at epithelial and synaptic junctions, and to play central roles in the formation of cell-cell junctions. The product of C. elegans lin-7 directly interacts with let-23 RTK and localize it at epithelial junctions. Here, we report three rat homologues of lin-7 ubiquitously expressed in various tissues. These homologues are accumulated at the junctional complex region in cultured Madin-Darby canine kidney cells, and are also localized at the synaptic junctions in neurons. The mammalian homologues of lin-7 may be implicated in the formation of cell-cell junctions.

Effect of polyphenon-60 on the development of renal cell tumors in rats treated with N-ethyl-N hydroxyethylnitrosamine.

Green tea consumed as a beverage in Asia contains polyphenols, which contain about a 15% mixture of catechins. The present paper reports the effect of polyphenon-60 (60% pure catechin) on the development of renal cell neoplasms in Wistar rats pretreated with N-ethyl-N-hydroxyethylnitrosamine (EHEN): 0.1% polyphenon-60 in block diet was given over a period of 30 weeks while EHEN was given in drinking water for 2 weeks. The results appears to show a tendency for green tea catechins (GTC) to decrease the incidence of renal cell tumors greater than 3 mm in diameter in Wistar rats but not tumors that are less than 3 mm in diameter. Polyphenon-60 did not affect EHEN initiation in the kidneys of rats. It is postulated that free radicals induced by EHEN may be suppressed by GTC, resulting in a lowering of the tendency for tumor growth.

Localization of membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 at tight junctions of epithelial cells.

Membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 and Synapse-associated protein (SAP) 97/human Discs-large tumor suppressor gene (hDLG) are ubiquitous isoforms of synaptic scaffolding molecule (S-SCAM) and Postsynaptic density (PSD)-95/SAP90, both of which are implicated in the structures of synapses, respectively. SAP97/hDLG is localized at epithelial junctions and may function as a scaffolding protein, but the subcellular localization or the function of MAGI-1/BAP1 has not been clarified. In intestinal epithelial cells, MAGI-1/BAP1 was localized at tight junctions, whereas SAP97/hDLG was localized diffusely at cell - cell junctions. In Madine Darby canine kidney (MDCK) cells, MAGI-1/BAP1 was colocalized with ZO-1, whereas SAP97/hDLG was colocalized with E-cadherin. In MDCK cells, dominant active and negative mutants of Rac1 small G protein changed the amounts of SAP97/hDLG at cell - cell junctions, but not that of MAGI-1/BAP1. When MDCK cells were switched to a low Ca2+ medium, E-cadherin disappeared from the plasma membrane, and cells were dissociated. The phorbol 12-myristate 13-acetate-treatment after the low Ca2+ switch induced a tight junction-like structure. MAGI-1/BAP1 was recruited with ZO-1 to this structure, but SAP97/hDLG or E-cadherin was not. These findings suggest that MAGI-1/BAP1 is a component of tight junctions of epithelial cells, and that its role is different from that of SAP97/hDLG.