Intracellular Ca2+ accumulation triggered by caffeine provokes resistance against a broad range of biotic stress in rice.

Chemical pesticides are still frequently overused to diminish such crop loss caused by biotic stress despite the threat to humans and the environment. Thus, it is urgent to find safer and more effective defense strategies. In this study, we report that caffeine, implanted through a transgenic approach, enhances resistance against variable biotic stresses in rice without fitness cost. Caffeine-producing rice (CPR) was generated by introducing three N-methyltransferase genes involved in the biosynthesis of caffeine in coffee plants. The CPR plants have no differences in morphology and growth compared to their wild-type counterparts, but they show strongly enhanced resistance to both bacterial leaf blight, rice blast, and attack of white-backed planthoppers. Caffeine acts as a repellent agent against rice pathogens. Moreover, caffeine triggers a series of Ca2+ signalling-like processes to synthesize salicylic acid (SA), a hormone associated with plant resistance. In CPR, phosphodiesterase was inhibited by caffeine, cAMP and cGMP increased, intracellular Ca2+ increased, phenylalanine lyase (PAL) was activated by OsCPK1, and SA synthesis was activated. This finding is a novel strategy to improve resistance against the biotic stresses of crops with a special type of defense inducer.

[Clinical Outcomes of Five Cases of Occult Breast Cancer in Which the Primary Site Was Not Detected in the Breast-by-Breast MRI].

Occult breast cancer is rare in practice. We studied the clinical outcomes of 5 occult breast cancers, including 2 with Luminal and 3 with non-Luminal subtypes, for which the primary site was not detected in the breast-by-breast MRI. The percentage of occult breast cancers that we encountered at our hospital was 0.11%. The mean age was 54 years. The Ki-67 labeling index value was 30% or higher for all the patients except one. Four patients were administered neoadjuvant chemotherapy and all but one patient received non-mastectomy and axillary dissection plus radiotherapy. We observed recurrent cases in one example each of the Luminal and HER2 subtypes, and both patients were less than 40 years old. The estimates of the probability of 5 year recurrence-free survival and 5 year overall survival were 40.0% and 66.7%, respectively. One recurrence case was a patient negative for ER and positive for HER2 wherein a breast cancer lesion appeared in the breast during post-treatment follow-up. Intrabreast relapse, which is itself rare in occult breast cancer, was observed 4 years postoperatively after standard treatment. Although there was no deviation according to subtype rate, the Ki-67 labeling index value was high and the prognosis was poor in our 5 cases.

Association between age factors and strategies for promoting participation in gastric and colorectal cancer screenings.

BACKGROUND: Despite the long history of cancer screening in Japan, the participation rates in gastric and colorectal cancer screenings have not increased. Strategies for improving the participation rates have been proposed, but differences in their effects among different age groups remain unclear. METHODS: The Japanese government conducted a national survey in all municipalities in Japan in 2010 to investigate whether the implementation of promotion strategies increased participation in cancer screening. We investigated the association between age factors and strategies for promoting participation in cancer screening based on this national survey. Multiple regression analysis with generalized linear model was performed using the participation rates in gastric and colorectal cancer screenings as dependent variables, and the following strategies for promoting participation as independent variables: 1) personal invitation letters, 2) household invitation letters, 3) home visits by community nurses, 4) screenings in medical offices, and 5) free cancer screening programs. RESULTS: One thousand six hundred thirty nine municipalities for gastric cancer screening and 1666 municipalities for colorectal cancer screening were selected for the analysis. In gastric and colorectal cancer screenings, the participation rates of individuals aged 60-69 years was higher than those of other age groups. Personal and household invitation letters were effective promotion strategies for all age groups, which encouraged even older people to participate in gastric and colorectal cancer screenings. Screening in medical offices and free screenings were not effective in all age groups. Home visits were effective, but their adoption was limited to small municipalities. CONCLUSIONS: To clarify whether promotion strategies can increase the participation rate in cancer screening among different age groups, 5 strategies were assessed on the basis of a national survey. Although personal and household invitation letters were effective strategies for promoting participation in cancer screening for all age groups, these strategies equally encouraged older people to participate in gastric and colorectal cancer screenings. If resource for sending invitation letters are limited, priority should be given to individuals who are in their 50s and 60s for gastric and colorectal cancer screening.

Safety and feasibility of adjuvant chemotherapy with S-1 in Japanese breast cancer patients after primary systemic chemotherapy: a feasibility study.

BACKGROUND: Advanced breast cancer patients have a higher risk of postoperative recurrence than early-stage breast cancer patients. Recurrence is believed to be caused by the increase in micrometases, which were not eradicated by preoperative or postoperative chemotherapy. Therefore, a new therapeutic strategy that can improve treatment efficacy is mandatory for advanced breast cancer. S-1 was shown to be effective and safe in Japanese metastatic breast cancer patients treated with previous chemotherapy, including anthracyclines. Thus, in this study, we evaluated S-1 as adjuvant chemotherapy in breast cancer patients after standard primary systemic chemotherapy. METHODS: The treatment consisted of 18 courses (a 2-week administration and a 1-week withdrawal; one year) administered at 80-120 mg/body/day. In cases judged to require postoperative radiotherapy, it was concurrently initiated on Day 1 of the study. If the estrogen receptor and/or human epidermal growth factor receptor 2 were positive, endocrine therapy and/or trastuzumab were permitted, concurrently. RESULTS: Of the 45 patients enrolled between September 2007 and September 2009 from 3 institutions, 43 patients were eligible. Thirty-two of the 43 (74.4%) patients received concurrent radiotherapy. Twenty-two of the 43 (51.2%) patients completed the scheduled courses of chemotherapy. The most common reasons for withdrawal of treatment were subjective symptoms, such as nausea, anorexia, or general fatigue during the first 9 courses of treatment in 9/43 (20.9%) patients, recurrence in 7/43 (16.3%) patients, and adverse events in 5/43 (11.6%) patients. The cumulative percentage of administration for 365 days was 66.4% (95% confidence interval: 50.8-79.1%). Although grade 3 neutropenia (9.3%), leukopenia (4.7%), and diarrhea (4.7%) were observed, they were manageable. No grade 4 adverse effects were observed. CONCLUSIONS: The percentage of Japanese breast cancer patients completing the 18-course treatment and the cumulative percentage of administration for 365 days using S-1 after standard primary systemic chemotherapy were similar with the results of another study of adjuvant chemotherapy for the Japanese gastric cancer patients with no severe adverse effects. A phase III trial investigating the usefulness of adjuvant S-1 is now ongoing in Japan, and it is expected that S-1 will have a significant survival benefit in breast cancer patients. UMIN000013469.

Marinibactrum halimedae gen. nov., sp. nov., a gammaproteobacterium isolated from a marine macroalga.

Phylogenetic and taxonomic characterization was performed for a bacterium, designated strain Q-192T, isolated from the surface of the green macroalga Halimeda sp., collected from the subtropical Ishigaki Island, Japan. The isolate was a polysaccharide-producing, Gram-stain-negative, aerobic, rod-shaped, motile bacterium with a polar flagellum. The isolate was slightly halophilic, required Na+, Mg2+ and Ca2+ ions for growth, but did not require growth factors. The only isoprenoid quinone was ubiquinone-8.The major cellular fatty acids were C18 : 1ω7c, C16 : 0 and C14 : 0. The main hydroxy fatty acid was C10 : 0 3-OH. The DNA G+C content was 45.9 mol%. Phylogenetic analysis of 16S rRNA gene sequences placed the isolate in the class Gammaproteobacteria. The phylogenetically closest relatives with validly published names were Pseudomaricurvus alkylphenolicus KU41GT, Teredinibacter turnerae T7902T, Pseudoteredinibacter isoporae SW-11T and Simiduia agarivorans SA1T with sequence similarities of 94.5, 94.1, 93.7 and 93.6 %, respectively. The isolate was distinguished from members of these genera by a combination of DNA G+C content, chemotaxonomic characteristics (respiratory quinone system, fatty acid profile and polar lipid composition) and other phenotypic features. Based on phylogenetic, genotypic, chemotaxonomic and phenotypic characteristics, strain Q-192T is considered to represent a novel species of a new genus, for which the name Marinibactrum halimedae gen. nov., sp. nov. is proposed. The type strain of Marinibactrum halimedae is Q-192T ( = NBRC 110095T = NCIMB 14932T).

Clinicopathological and prognostic impact of imaging of breast cancer angiogenesis and hypoxia using diffuse optical spectroscopy.

Near-infrared diffuse optical spectroscopy (DOS) imaging can non-invasively measure tumor hemoglobin concentration using high contrast to normal tissue, thus providing vascularity and oxygenation status. We assessed the clinical usefulness of DOS imaging in primary breast cancer. In all, 118 women with a histologically confirmed diagnosis of primary malignant tumor were enrolled. All participants underwent testing using time-resolved DOS before treatment initiation. Visual assessment of DOS imaging for detecting tumors was carried out by two readers blinded to the clinical data. Relative total hemoglobin (rtHb) and oxygen saturation (stO2 ) of the tumors was compared with clinicopathological variables and 10-year prognosis was calculated. Sensitivity for detecting a tumor based on the rtHb breast map was 62.7% (74/118). The sensitivity depended on T stage: 100% (7/7) for T3, 78.9% (45/57) for T2, 44.7% (17/38) for T1, and 31.3% (5/16) for Tis . Tumors showed unique features of higher rtHb with a wider range of stO2 than normal breast tissue, depending on histological type. There was a significant correlation of rtHb with tumor size, lymphatic vascular invasion, and histological grade, and of stO2 with age and tumor size. Neither rtHb nor stO2 correlated with intrinsic biomarkers such as estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2; rtHb inversely correlated with 10-year relapse-free survival and overall survival, with statistical significance. Diffuse optical spectroscopy imaging has limited utility for the early detection of breast cancer; nonetheless, the findings suggest that the degree of tumor angiogenesis and hypoxia may be associated with tumor aggressiveness and poor prognosis.

Contribution of bone marrow-derived mesenchymal stem cells to the morphological changes in the bladder after partial outlet obstruction: a preliminary study.

OBJECTIVES: To investigate the contribution of bone marrow-derived mesenchymal stem cells to the changes in bladder morphology in response to partial bladder outlet obstruction. METHODS: Allogenic bone marrow cells were transplanted from transgenic rats expressing green fluorescent protein into female Sprague-Dawley rats 1 day after their bone marrow-derived mesenchymal stem cells had been destroyed by irradiation. This generated chimeric rats in which green fluorescent protein labeled bone marrow-derived mesenchymal stem cells replaced host bone marrow-derived mesenchymal stem cells. The animals received partial bladder outlet obstruction or sham surgery 6 weeks later. The animals were killed 6 weeks after the surgery, and bladder tissue was prepared for immunofluorescence with antibodies against a urothelium marker (AE1/AE3), a myofibroblast marker (vimentin), a smooth muscle marker and green fluorescent protein. RESULTS: More labeled bone marrow-derived mesenchymal stem cells were found in the partial bladder outlet obstruction group than in the in the sham group. Most bone marrow-derived mesenchymal stem cells were present around the basement membrane (laminin) and lamina propria below the urothelium. Bone marrow-derived mesenchymal stem cells were also found in the urothelium layer, and some of them were double-stained with green fluorescent protein and AE1/AE3. Some bone marrow-derived mesenchymal stem cells, which were located in the interstitial tissue, were double-stained with green fluorescent protein and vimentin. Bone marrow-derived mesenchymal stem cells, which migrated into the smooth muscle layer, showed fusiform morphology, and some were double-stained with green fluorescent protein and smooth muscle marker. CONCLUSIONS: Bone marrow-derived mesenchymal stem cells home to the partial bladder outlet obstruction bladder, and these cells have the potential to differentiate into the several components of bladder tissue including the urothelium, myofibroblasts and smooth muscle cells. Thus, bone marrow-derived mesenchymal stem cells contribute to the morphological changes of the bladder in response to partial bladder outlet obstruction.

Optical imaging of tumor vascularity associated with proliferation and glucose metabolism in early breast cancer: clinical application of total hemoglobin measurements in the breast.

BACKGROUND: Near-infrared optical imaging targeting the intrinsic contrast of tissue hemoglobin has emerged as a promising approach for visualization of vascularity in cancer research. We evaluated the usefulness of diffuse optical spectroscopy using time-resolved spectroscopic (TRS) measurements for functional imaging of primary breast cancer. METHODS: Fifty-five consecutive TNM stage I/II patients with histologically proven invasive ductal carcinoma and operable breast tumors (<5 cm) who underwent TRS measurements were enrolled. Thirty (54.5%) patients underwent 18F-fluoro-deoxy-glucose (FDG) positron emission tomography with measurement of maximum tumor uptake. TRS was used to obtain oxyhemoglobin, deoxyhemoglobin, and total hemoglobin (tHb) levels from the lesions, surrounding normal tissue, and contralateral normal tissue. Lesions with tHb levels 20% higher than those present in normal tissue were defined as "hotspots," while others were considered "uniform." The findings in either tumor type were compared with clinicopathological factors. RESULTS: "Hotspot" tumors were significantly larger (P= 0.002) and exhibited significantly more advanced TNM stage (P=0.01), higher mitotic counts (P=0.01) and higher levels of FDG uptake (P=0.0004) compared with "uniform" tumors; however, other pathological variables were not significantly different between the two groups. CONCLUSIONS: Optical imaging for determination of tHb levels allowed for measurement of tumor vascularity as a function of proliferation and glucose metabolism, which may be useful for prediction of patient prognosis and potential response to treatment.

Pulmonary tumor thrombotic microangiopathy showing aggressive course after transurethral resection of urinary bladder: an autopsy case report.

A 77-year-old man developed pulmonary tumor thrombotic microangiopathy (PTTM) 2 days after undergoing transurethral resection for urothelial carcinoma (G3) of the urinary bladder and died of respiratory failure 6 days later. Histological findings demonstrated marked intimal fibrocellular proliferation, fibrin thrombi, and both cancer cells and fibrin thrombi in the arteries of the lungs, findings consistent with PTTM. Prominent stenosis in arteries smaller than 300 µm was also seen. The Ki-67 labeling index of primary and metastasized cancer cells was 62.4 % and 70.2 %, respectively. The membranes of metastasized cancer cells expressed E-cadherin, similar to membranes in the urinary bladder. An aggressive PTTM course is affected by intimal fibrocellular proliferation and the high cell proliferation of cancer cells. Furthermore, prominent stenosis in small arteries and membranous staining of E-cadherin of metastasized cells suggest that cancer cells formed clusters by maintaining adhesion molecules and migrated into the arteries of the lungs, where they easily caused damage to the endothelium of small arteries, in contrast to dispersed cancer cells.

Pathological characteristics and clinical course of bladder tumour developing after nephroureterectomy.

OBJECTIVES: To determine the pathological features and clinical course of intravesical recurrence after nephroureterectomy (NU) for upper urinary tract (UUT) cancer. PATIENTS AND METHODS: Among 325 patients undergoing NU with bladder cuff excision for UUT cancer, in this retrospective multi-institutional study we evaluated 113 who developed bladder tumour after NU. Excluding patients with (i) perioperative systemic chemotherapy or radiotherapy for UUT cancer; (ii) a history of previous or synchronous bladder cancer at the time of NU; (iii) distant metastasis at the time of NU; (iv) a follow-up of <1 year after the initial bladder cancer recurrence; or (v) missing data, 74 patients were included in this study. We compared the pathology between UUT cancer and the first bladder cancer recurrence, using Fisher's exact test. Further intravesical recurrence and bladder cancer progression was analysed using the Kaplan-Meier method, with the log-rank test used to assess significance. A Cox proportional hazard model was used for multivariate analysis. RESULTS: The grade of the first bladder cancer recurrence strongly correlated with that of the UUT tumour (P < 0.001) and the carcinoma in situ (CIS) lesion with the first bladder cancer recurrence correlated with high grade (grade 3) UUT tumour (P < 0.001). In all, 56 of the assessable 70 patients further developed intravesical recurrence at a median interval of 7 months after the first bladder cancer recurrence. There were no clinicopathological factors that predicted the second recurrence. Progression occurred in 14 patients, at a median interval of 25 months. A CIS lesion with the first bladder cancer recurrence was a risk factor for progression on multivariate analysis. CONCLUSIONS: A large proportion of the patients who developed bladder tumour after NU had further intravesical recurrence, which indicated its refractory nature. Especially when a CIS lesion is detected in the initial intravesical recurrence, a careful follow-up is mandatory to detect bladder cancer progression.

BiClamp forceps significantly shorten the operation time for breast surgery.

PURPOSE: A novel approach was introduced for breast surgery using the BiClamp, a new bipolar thermal energy device, to avoid complications and to shorten the time required for the dissection of the axillary lymph nodes. METHODS: Thirty-six patients with early breast cancer were assessed. The surgical parameters were compared between the procedures performed using the BiClamp technique (n = 14) and conventional surgery with suture ligation (n = 22). The parameters included the operation time, blood loss, and discharge on the first postoperative day. In addition, each of those parameters was compared between the patients with a high body mass index (BMI) (>22) and a low BMI (< or =22). The sealed vessels were examined histologically and heat-associated morphological vessel wall alterations were evaluated. RESULTS: The operation time was significantly shorter in the BiClamp group than in the control group (P = 0.017, 90 +/- 18 vs 115 +/- 33 min). In addition, the blood loss in the BiClamp group tended to be smaller than in the control group, but the difference was not significant (P = 0.54, 61 +/- 47 vs 74 +/- 67 g). No other parameters showed any significant differences between the two groups. CONCLUSION: The BiClamp thermofusion technique was safe and useful in breast surgery involving axillary dissection.

Polyamines as a common source of hydrogen peroxide in host- and nonhost hypersensitive response during pathogen infection.

The hypersensitive response (HR) is a powerful resistance system that plants have developed against pathogen attack. There are two major pathways for HR induction; one is through recognition of the pathogen by a specific host protein, and is known as the host HR. The other is through common biochemical changes upon infection--the nonhost HR. We previously demonstrated that hydrogen peroxide derived from polyamine degradation by polyamine oxidase triggers the typical host HR in tobacco plants upon infection with tobacco mosaic virus. However, it remains to be determined whether or not polyamines are involved in the nonhost HR in tobacco, and in the host HR in other plant species. When tobacco plants were infected with Pseudomonas cichorii, a representative nonhost pathogen, transcripts for six genes encoding enzymes for polyamine metabolism were simultaneously induced, and polyamines were accumulated in apoplasts. Hydrogen peroxide was concomitantly produced and hypersensitive cell death occurred at infected sites. Silencing of polyamine oxidase by the virus-induced gene silencing method resulted in suppression of hydrogen peroxide production and in disappearance of visible hypersensitive cell death with an increase in bacterial growth. Our results indicated that polyamines served as the source of hydrogen peroxide during the nonhost HR in tobacco plants. Further analysis revealed that polyamines were accumulated in apoplasts of Arabidopsis thaliana infected with Pseudomonas syringae, and of rice infected with Magnaporthe grisea, both causing the typical host HR. As in tobacco, it is conceivable that the same mechanism operates for nonhost HR in these plants. Our present observations thus suggested that polyamines are commonly utilized as the source of hydrogen peroxide during host- and nonhost HRs in higher plants.

Improvement of the light extraction efficiency of top-emitting organic light-emitting diodes by a two-dimensional diffraction layer fabricated using self-assembled nanoparticles.

The light extraction efficiency of top-emitting organic light-emitting diodes (OLEDs) was improved by insertion of a two-dimensional (2D) diffraction layer. The 2D diffraction layer was fabricated by our original nanofabrication technique, the embedded particle monolayer method, which could form a self-assembled particle monolayer. As a result, the electroluminescence intensity of the device with the 2D diffraction layer was improved by 1.67 times (in total luminous flux) and 2.07 times (in peak wavelength). High luminance top-emitting OLEDs were fabricated using the potentially low-cost self-assembling technique.

Effect of a test dentifrice containing nano-sized calcium carbonate on remineralization of enamel lesions in vitro.

The efficacy of a test dentifrice containing nano-sized (several tens to hundreds of nm) calcium carbonate (hereafter NC) on enamel lesion remineralization was studied in an in vitro system that employed collagen-coated wells for cell culture, as a model of oral surfaces for NC retention. The well surfaces were treated with the test dentifrice and briefly rinsed with distilled water. Thin sections of enamel with artificial subsurface demineralization were remineralized in the plate wells containing remineralizing solution. The dentifrice treatment was repeated twice a day (in the morning and evening) for 20 days. After remineralization, microradiographic analysis was performed to evaluate the rate of lesion remineralization on the sections. The test dentifrice showed a statistically significant mineral gain (48.8% decrease in DeltaZ % x microm from the baseline value), indicating lesion remineralization, whereas the placebo dentifrice without NC did not. An elevated Ca concentration in the remineralizing solution was also observed after a single treatment with the test dentifrice. We conclude that the test dentifrice has potential to remineralize incipient enamel lesions due to the unique properties of NC, which is retained on oral surfaces, thereafter releasing Ca ions into oral fluids (saliva, plaque).

One-year clinical outcomes of anticoagulation therapy among Japanese patients with atrial fibrillation: The Hyogo AF Network (HAF-NET) Registry.

BACKGROUND: Although anticoagulation therapy could reduce the risk of strokes in patients with atrial fibrillation (AF), large-scale investigations in the direct oral anticoagulant (DOAC) and AF catheter ablation (CA) era are lacking. METHODS: This study was designed as a prospective, multicenter, observational study and a total of 2113 patients from 22 institutions were enrolled in the Hyogo area. RESULTS: The mean age and CHADS2 score were 70.1 ± 10.8 years old and 1.5 ± 1.1, respectively. The follow-up period was 355 ± 43 days. CA was performed in 614 (29%) and DOACs were prescribed in 1118 (53%) patients. Ischemic strokes/systemic embolisms (SEs) and major bleeding occurred in 13 (0.6%) and 17 (0.8%) patients, respectively. New onset dementia, hospitalizations for cardiac events, and all-cause death occurred in eight (0.4%), 60 (2.8%), and 29 (1.4%) patients, respectively. A multivariate analysis demonstrated that persistent AF and the body weight (BW) were associated with ischemic strokes/SEs and major bleeding, respectively (persistent AF: hazard ratio, 9.57; 95%CI, 1.2-74.0; P = .03; BW: hazard ratio, 0.94; 95%CI, 0.90-0.99; P = .02). AFCA history was associated with the cardiac events (hazard ratio, 0.44; 95%CI, 0.20-0.99; P = .04). Age was associated with new onset dementia (hazard ratio, 1.1; 95%CI, 1.0-1.2; P = .03). CONCLUSIONS: In the DOAC and CA era, the incidence of ischemic strokes/SEs, major bleeding and cardiac events could be dramatically reduced in patients with AF. However, some unsolved issues of AF management still remain especially in elderly patients with persistent AF and a low BW.

Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering.

Details of the recently elucidated biosynthetic pathways of caffeine and related purine alkaloids are reviewed. The main caffeine biosynthetic pathway is a sequence consisting of xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine. Genes encoding N-methyltransferases involved in three of these four reactions have been isolated and the molecular structure of N-methyltransferases investigated. Pathways for the catabolism of caffeine have also been studied, although there are currently no reports of enzymatic and genetic studies having been successfully carried out. Metabolism of purine alkaloids in species including Camellia, Coffea, Theobroma and Ilex plants is summarised, and evidence for the involvement of caffeine in chemical defense and allelopathy is discussed. Finally, information is presented on metabolic engineering that has produced coffee seedlings with reduced caffeine content, and transgenic caffeine-producing tobacco plants with enhanced disease resistance.

Pathogen resistance of transgenic tobacco plants producing caffeine.

Caffeine (1,3,7-trimethylxanthine) is a typical purine alkaloid, and produced by a variety of plants such as coffee and tea. Its physiological function, however, is not completely understood, but chemical defense against pathogens and herbivores, and allelopathic effects against competing plant species have been proposed. Previously, we constructed transgenic tobacco plants, which produced caffeine up to 5 microg per gram fresh weight of leaves, and showed them to repel caterpillars of tobacco cutworms (Spodoptera litura). In the present study, we found that these transgenic plants constitutively expressed defense-related genes encoding pathogenesis-related (PR)-1a and proteinase inhibitor II under non-stressed conditions. We also found that they were highly resistant against pathogens, tobacco mosaic virus and Pseudomonas syringae. Expression of PR-1a and PR-2 was higher in transgenic plants than in wild-type plants during infection. Exogenously applied caffeine to wild-type tobacco leaves exhibited the similar resistant activity. These results suggested that caffeine stimulated endogenous defense system of host plants through directly or indirectly activating gene expression. This assumption is essentially consistent with the idea of chemical defense, in which caffeine may act as one of signaling molecules to activate defense response. It is thus conceivable that the effect of caffeine is bifunctional; direct interference with pest metabolic pathways, and activation of host defense systems.

Functional diversification of a basic helix-loop-helix protein due to alternative transcription during generation of amphidiploidy in tobacco plants.

A plastid-resident basic helix-loop-helix protein, previously identified in Nicotiana tabacum and designated as NtWIN4 (N. tabacum wound-induced clone 4), has been converted from a nuclear transcription repressor into a plastid-resident regulatory factor through replacement of the DNA-binding domain with a plastid transit sequence during evolution. N. tabacum is a natural amphidiploid plant derived from Nicotiana tomentosiformis and Nicotiana sylvestris and immunoblot staining using anti-NtWIN4 antibodies identified two protein species, a 26 kDa form and a 17 kDa form, in N. sylvestris, whereas only the 17 kDa form was found in N. tabacum. The 26 kDa protein is produced when translation starts from the first AUG codon of the mRNA and is predominantly localized in the cytoplasm and nucleus, whereas the 17 kDa protein is derived from a 24 kDa precursor protein, synthesized from the second AUG codon, and localizes only to plastids. Subsequent analyses revealed that the lengths of the mRNAs vary in the two plant species. One major form lacks the first AUG, while minor populations possess variable 5'-untranslated regions prior to the first AUG codon. Translation of the two types produces the 24 kDa and 26 kDa proteins respectively. In vitro translation assays indicated that initiation frequency from the first AUG codon is higher in mRNAs from N. sylvestris than from N. tabacum. In contrast, initiation from the second AUG codon was found to be equally efficient in mRNAs from both species. These results suggest that both mRNA populations and translation efficiency changed during the amphidiploidization responsible for generation of N. tabacum. This scheme could reflect a molecular mechanism of protein evolution in plants.

Effect of a xylitol and fluoride containing toothpaste on the remineralization of human enamel in vitro.

The aim of this study was to examine the effect of a fluoride and xylitol containing toothpaste on the remineralization of human enamel using Quantitative Light-induced Fluorescence (QLF). Human extracted teeth were cut longitudinally into three or four parts, and artificial subsurface lesion windows (2 mm x 3 mm) were created by immersion in demineralizing solution. Each enamel sample (n = 7) was treated for 3 min at 25 degrees C twice a day for consecutive 14 days with the slurry of a silica-based toothpaste 1) without F- and xylitol (blank), 2) with 500 ppm F- and 3) with 500 ppm F- and 5% xylitol toothpaste. In addition, we measured the remineralization ability of a commercially available toothpaste 4) with 500 ppm F-. The average fluorescence loss F (%) and lesion size (mm2) were determined with QLF. Another variable, delta Q, which was defined as the fluorescence loss integrated over the lesion size (%, mm2), was calculated. The results showed that the combination of 500 ppm F- and 5% xylitol toothpaste significantly (P < 0.05) recovered both the size and delta Q compared to the other groups. These findings suggested that inclusion of xylitol in fluoride toothpaste might be useful to enhance the remineralization in vivo.

Knee Motion Generation Method for Transfemoral Prosthesis Based on Kinematic Synergy and Inertial Motion.

Previous research has shown that the effective use of inertial motion (i.e., less or no torque input at the knee joint) plays an important role in achieving a smooth gait of transfemoral prostheses in the swing phase. In our previous research, a method for generating a timed knee trajectory close to able-bodied individuals, which leads to sufficient clearance between the foot and the floor and the knee extension, was proposed using the inertial motion. Limb motions are known to correlate with each other during walking. This phenomenon is called kinematic synergy. In this paper, we measure gaits in level walking of able-bodied individuals with a wide range of walking velocities. We show that this kinematic synergy also exists between the motions of the intact limbs and those of the knee as determined by the inertial motion technique. We then propose a new method for generating the motion of the knee joint using its inertial motion close to the able-bodied individuals in mid-swing based on its kinematic synergy, such that the method can adapt to the changes in the motion velocity. The numerical simulation results show that the proposed method achieves prosthetic walking similar to that of able-bodied individuals with a wide range of constant walking velocities and termination of walking from steady-state walking. Further investigations have found that a kinematic synergy also exists at the start of walking. Overall, our method successfully achieves knee motion generation from the initiation of walking through steady-state walking with different velocities until termination of walking.