Blood group B gene is barely expressed in in vitro erythroid culture of Bm-derived CD34+ cells without an erythroid cell-specific regulatory element.

BACKGROUND AND OBJECTIVES: Previously, a weak phenotype Am or Bm was assumed to be caused by a reduction of A or B gene expression in bone marrow cells, but not in mucus-secreting cells. However, ABO expression has not been examined in erythroid progenitor cells of Am or Bm individuals. MATERIALS AND METHODS: We carried out in vitro erythroid differentiation of CD34(+) cells from peripheral blood of a Bm individual harbouring a 3.0-kb deletion including an erythroid cell-specific regulatory element, named the +5.8-kb site, in intron 1 of the human ABO blood group gene. RESULTS: During the in vitro differentiation of CD34(+) cells from this Bm individual into erythroid cells, B-antigens were not detectable on the cultured cells by flow cytometric analysis, and allele-specific RT-PCR consistently detected the transcripts from the O allele, but not from the B allele. Moreover, chromatin immunoprecipitation assay demonstrated that both RUNX1 and GATA-2 or GATA-1 were bound to the +5.8-kb site in cultured erythroid cells expressing ABO. CONCLUSION: It is likely that the +5.8-kb site enhances transcription from the ABO promoter in erythroid cells through binding of RUNX1 and GATA-2 or GATA-1.

Parallel comparison of apheresis-collected platelet concentrates stored in four different additive solutions.

BACKGROUND AND OBJECTIVES: Partially replacing plasma with additive solutions in platelet (PLT) concentrates (PCs) may help to reduce transfusion reactions. Constituents of PLT additive solutions (PASs) have been revealed to affect the quality of PCs. Previous studies involved pairwise comparison of identical PLTs with two different PASs or multicomparison using random PLTs with three or more PASs. In this study, we performed parallel comparison using PCs from identical donors with four PASs. In addition to traditional parameters, the release of bioactive substances and plasma proteins was assessed. MATERIALS AND METHODS: Platelets collected four times by apheresis from three donors were suspended in Intersol, SSP+, Composol or M-sol with 35% autologous plasma. The PC parameters, including PLT activation markers, glucose consumption, chemokines and plasma proteins, were assessed during 5-day storage. RESULTS: Mean PLT volumes were decreased in SSP+, Composol and M-sol after 5-day storage, with significant differences, whereas the hypertonic shock response (HSR) was decreased only in Intersol. Glucose consumption was faster in Intersol and M-sol than in SSP+ or Composol. PLT activation, determined as CD62P, sCD62P, sCD40L and RANTES, was significantly higher in Intersol than the other three PASs. No marked change was observed in fibrinopeptide A and C3a in any PASs. CONCLUSIONS: M-sol, SSP+ and Composol effectively preserved the quality of PCs. PLT activation was significantly enhanced in Intersol compared with the other three PASs. These effects seem to depend on magnesium and potassium as a constituent. Parallel comparison further verified that the PC quality largely depended on PASs but not donors.

Development of a new aortoscope system for the use of endovascular intervention.

"Balloon aortoscopy" is a technique for viewing inner wall of aorta and used in clinics. By this method, endoluminal aortic surface could be clearly monitored, however, during this period, the aortic blood flow is blocked off by the inflated balloon. To solve this clinical problem, we have been developing a prototype aortoscope system without blocking off aortic flow aiming for the use of an assistive technique for endovascular interventions such as stent-graft placement for aortic aneurysm and have been evaluating through in vitro and in vivo tests. The technique introduced for this purpose was the use of intermittent and instantaneous saline jet controlled by a high-speed electromagnetic valve synchronized to heart beat (diastolic phase). In the previous study, we designed an endoscope with two channels (one for saline discharge and the other for forceps insertion), and confirmed the validity of this method by in vitro and in vivo tests. Based on these findings, in this study, we have newly designed a conventional and low price endoscope system aiming for wide clinical use. From the results of in vitro tests using a mock circulation system, it was confirmed that the newly designed system was capable of visualizing a target installed on an inner surface of the mock system suggesting an availability of the system for an aortoscope without blocking off aortic flow.

Evaluation of a newly designed endoscope for observing inner wall of large arteries for the use of endovascular intervention.

A prototype endoscope for observing inner wall of large arteries was specially designed and evaluated through in vitro and in vivo tests. The purpose of this endoscope is to visualize the inner wall of large arteries, e.g., an aorta, without blocking off the blood stream aiming for the use of an assistive technique for endovascular interventions such as stent-graft placement for aortic aneurysm. The technique newly introduced for this purpose was the use of intermittent high-pressure saline jet synchronized to heart beat (diastolic phase). In the previous studies using commercially available bronchoscopes, we confirmed the validity of the system utilizing this technique [1, 2]. Based on these findings, in this study, we have specially designed a new endoscope with two channels, one for saline discharge and the other for forceps, and evaluated its performance through in vitro and in vivo tests. From the results of in vitro tests using a mock circulation system, it was confirmed that the newly designed endoscope was capable of visualizing a target installed on an inner surface of the mock system. Also confirmed through in vivo tests using swine was that we could observe bifurcation in descending aorta, e.g., left renal artery, without stopping off the blood stream.

A new proposal of tailored bioinstrumentation using rapid prototyping and three-dimensional CAD--first trial to develop individually designed cuff-units for continuous blood pressure measurement.

The concept of tailored bioinstrumentation using rapid prototyping and three-dimensional CAD (3D-CAD) was proposed. This concept is to make individually designed and fabricated sensor unit to attach human body. Within the proposed concept, cuff-units for continuous blood pressure measurement were individually designed using 3D-CAD and fabricated automatically. As the result, blood pressure wave forms can be obtained using the finally developed cuff units. Using rapid prototyping device, the design and fabrication process were accelerated without any artisan-like high skilled persons.

Haemostatic effects of polymerized albumin particles carrying fibrinogen gamma-chain dodecapeptide as platelet substitutes in severely thrombocytopenic rabbits.

Our purpose was to produce a platelet substitute that could enhance haemostatic ability using rabbits with severe thrombocytopenia. We have developed polymerized albumin particles (polyAlb) for treatment of bleeding and focused on a dodecapeptide, HHLGGAKQAGDV (H12), as a useful ligand for activated platelet. This sequence occurs only at the carboxy-terminus of the fibrinogen gamma-chain (gamma 400-411). H12 was conjugated to the surface of polyAlb modified with poly(ethylene glycol) (PEG) chains to produce blood-compatible particles (H12-PEG-polyAlb) that had prolonged blood residence time and enhanced stability in vitro and in vivo. The H12-PEG-polyAlb was administered intravenously to rabbits with severe thrombocytopenia, and the ear bleeding time was measured in order to evaluate the haemostatic effect. The H12-PEG-polyAlb significantly shortened the ear bleeding time of severely thrombocytopenic rabbits and showed no effect on the inhibition or promotion of endogenous and exogenous coagulation activities. Furthermore, we could assess the haemostatic capacity of the H12-PEG-polyAlb, based on the relationship between transfused platelet count and the bleeding time. The H12-PEG-polyAlb may be a suitable candidate for an alternative to human platelet concentrates infused to treat bleeding in patients with severe thrombocytopenia.

Feasibility study on driver's stress detection from differential skin temperature measurement.

Prolonged monotonous driving may lower a driver's awareness level as well as increasing their stress level due to the compulsion to maintain safe driving, which may result in an increased risk of a traffic accident. There is therefore an opportunity for technological assessment of driver physiological status to be applied in-car, hopefully reducing the incidence of potentially dangerous situations. As part of our long-term aim to develop such a system, we describe here the investigation of differential skin temperature measurement as a possible marker of a driver's stress level. In this study, healthy male (n=18) & female (n=7) subjects were investigated under environment-controlled conditions, whilst being subjected to simulated monotonous travel at constant speed on a test-course. We acquired physiological variables, including facial skin temperature which consists of truncal and peripheral skin temperatures (Ts) using thermography, beat-by-beat blood pressure (BP), cardiac output (CO), total peripheral resistance (TPR), and normalized pulse volume (NPV) used as an indicator of local peripheral vascular tone. We then investigated the driver's reactivity in terms of skin temperatures with this background of cardiovascular haemodynamics. We found that the simulated monotonous driving produced a gradual drop in peripheral Ts following the driving stress, which, through interpretation of the TPR and NPV recordings, could be explained by peripheral sympathetic activation. On the other hand, the truncal Ts was not influenced by the stress. These findings lead us to suggest that truncal-peripheral differential Ts could be used as a possible index indicative of the driver's stress.

A preliminary study on driver's stress index using a new method based on differential skin temperature measurement.

Prolonged periods of driving in monotonous situations may lower a driver's activation state as well as increasing their stress level due to the compulsion to maintain safe driving, which may result in an increased risk of a traffic accident. There is therefore an opportunity for technological assessment of driver physiological status to be applied in-car, hopefully reducing the incidence of potentially dangerous situations. As part of our long-term aim to develop such a system, we describe here the investigation of differential skin temperature measurement as a possible marker of a driver's stress level. 10 healthy male subjects were studied, under environment-controlled conditions, whilst being subjected to simulated monotonous travel at constant speed on a test-course. We acquired measurements of relevant physiological variables, including truncal and peripheral skin temperatures (T(s)), beat-by-beat blood pressure (BP), cardiac output (CO), total peripheral resistance (TPR), and normalized pulse volume (NPV) used as an indicator of local peripheral vascular tone. We then investigated the driver's reactivity in terms of cardiovascular haemodynamics and skin temperatures. We found that the simulated monotonous driving produced a gradual drop in peripheral T(s) following the driving stress, which, through interpretation of the TPR and NPV recordings, could be explained by peripheral sympathetic activation. On the other hand, the truncal T(s) was not influenced by the stress. These findings lead us to suggest that truncal-peripheral differential T(s) might be used as a possible index indicative of the driver's stress.

Development of a fully automated network system for long-term health-care monitoring at home.

Daily monitoring of health condition at home is very important not only as an effective scheme for early diagnosis and treatment of cardiovascular and other diseases, but also for prevention and control of such diseases. From this point of view, we have developed a prototype room for fully automated monitoring of various vital signs. From the results of preliminary experiments using this room, it was confirmed that (1) ECG and respiration during bathing, (2) excretion weight and blood pressure, and (3) respiration and cardiac beat during sleep could be monitored with reasonable accuracy by the sensor system installed in bathtub, toilet and bed, respectively.

A new non-invasive method for measuring blood glucose using instantaneous differential near infrared spectrophotometry.

We describe further development of a novel method for non-invasive measurement of blood glucose concentration (BGL), named Pulse Glucometry, based on differential near infrared spectrophotometry. Sequential temporal differences of infrared transmittance spectra from the radiation intensity (I(lambda)) emerging from a fingertip containing an arterial pulse component (DeltaI(lambda)) are analysed. To perform the measurements we developed a new high-speed spectrophotometer, covering the wavelength range from 900 to 1700 nm, scanning at a maximum spectral rate of 1800 spectra/s, with a minimum exposure time of 20 micros. Spectra related only to the pulsatile blood component are derived, thus minimising influences of basal components such as resting blood volume, skin, muscle and bone. We have now improved the performance of the spectrophotometer and in the present paper we describe new in vivo measurements carried out in 23 healthy volunteers undergoing glucose tolerance tests. Blood samples were collected from the cephalic vein simultaneously with radiation intensity measurements in the fingertip every 10 min before and after oral administration of glucose solution for 120 min. BGL values were then predicted using a PLS calibration model and compared with blood values determined by colorimetric assay. The precision and accuracy of the non-invasive determinations are encouraging.

A third-generation bisphosphonate, minodronic acid (YM529), successfully prevented the growth of bladder cancer in vitro and in vivo.

Minodronic acid (YM529) is a third-generation bisphosphonate (BP) that has been shown to directly and indirectly prevent proliferation, induce apoptosis, and inhibit metastasis of various types of cancer cells. In this study, we have investigated the therapeutic efficacy of YM529 against bladder cancer, both in vitro and in vivo. YM529 inhibited geranylgeranylation as well as farnesylation and reduced the growth of all seven bladder cancer cell lines in a dose- and time-dependent manner in vitro. YM529 demonstrated a good synergistic or additive antiproliferative effect when administered in combination with cisplatin or paclitaxel. Immunohistochemical study revealed YM529 inhibited the prenylation of Rap1A in vivo. YM529 administered systemically did not markedly inhibit the growth of visceral metastases but it showed a significant anticancer effect on bone metastases monitored by an in vivo imaging system. Moreover, intravesical YM529 demonstrated significant growth inhibition in a bladder cancer orthotopic model. No adverse effects were associated with the systemic as well as the intravesical treatment regimens. In conclusion, our study suggests that YM529 may be a potent anticancer agent for bladder cancer. The efficacy and safety of this BP as an agent for combination chemotherapies against bladder cancer should be verified by early-phase clinical trials.

Cloning, functional expression and promoter analysis of xylanase III gene from Trichoderma reesei.

In this study, the xyn3 gene from the filamentous mesophilic fungus Trichoderma reesei (Hypocrea jecorina) PC-3-7 was cloned and sequenced. Analysis of the deduced amino acid sequence of XYN III revealed considerable homology with xylanases belonging to glycoside hydrolase family 10. These results show that XYN III is distinguishable from XYN I and XYN II, two other T. reesei xylanases that belong to the glycosidase family 11. When xyn3 was expressed in Escherichia coli, significant activity was observed in the cell-free extract, and higher activity (13.2 U/ml medium) was recovered from the inclusion bodies in the cell debris. The sequence of the 5'-upstream region of the gene in the parent strain QM9414 is identical to that of PC-3-7, although the expression level of xyn3 in PC-3-7 has been reported to be at least 1,000 times greater than in QM9414. These results suggest that xyn3 expression in T. reesei QM9414 is silenced. The consensus sequences for ACEI, ACEII, CREI, and the Hap2/3/5 protein complex are all present in the upstream region of xyn3. Deletion analysis of the upstream region revealed that two regions containing consensus sequences for the known regulatory elements play important roles for xyn3 expression.

Hemodynamic responses during simulated automobile driving in a monotonous situation.

Long hours of automobile driving under monotonous situations may cause the lowering of what we term a driver's activation state (DAS) or in other words the production of drowsiness, resulting in an increased risk of a traffic accident. There is therefore a need to create a newly advanced system focused on the DAS in-car, hopefully thus avoiding potentially dangerous situations. In order to develop such a system as a final goal, we have firstly set out to acquire such cardiovascular variables as beat-by-beat blood pressure (BP), RR interval from ECG and normalized pulse volume (NPV) used as a peripheral vascular tone of alpha-adrenergic sympathetic activity, during presentation to the driver of a screen movie simulating monotonous travel at constant speed on a test-course. Subsequently, we have investigated the reactivity in terms of the driver's cardiovascular hemodynamics. Through the successful monitoring of cardiovascular parameters during the movie presentation obtained in 11 healthy male subjects, the following results were obtained: The monotonous driving produces a statistically significant gradual rise in BP following drowsiness, which could be explained by enhancement of sympathetic activity using a time-frequency analysis of BP and RR. This finding strongly indicates that continuous driving in such monotonous situations can make a driver considerably stressful and thus may cause a gradual increase in BP, and that this gradual BP increase may be used as a possible index relevant to the DAS. This finding was also confirmed by the analysis of NPV, suggesting that the gradual increase in BP during the monotonous driving would be rather caused by a regulation of peripheral vasomotor constriction.

Development of an ambulatory device for monitoring posture change and walking speed for use in rehabilitation.

Monitoring of posture change in sagittal plane and walking speed is important for evaluate the effectiveness of rehabilitation program or brace. We have developed a wearable device for monitoring human activity. However, in the previous system, there still remain several drawbacks for practical use such as accuracy in angle measurement, cumbersome cable arrangements, and so on. In order to improve these practical drawbacks, a new sensor system was designed, and its availability was evaluated. The results demonstrated that the accuracy of this system showed superior to that of the previous, and this system appears to be a significant means for quantitative assessment of the patient's motion.

Assessing the Effectiveness of Increased FIO2for Enhancing Driver's Activation State Using Simulated Monotonous Driving.

Lowering of what we term a driver's Activation State (AS) during monotonous driving conditions may increase the risk of an accident. To develop an in-car environment that allows active driving - "Biofee dforward System" - we have investigated the effects of applying a stimulus of increased inspired oxygen fraction (FIO2) supply on a driver's AS, using simulated monotonous driving. We used our previously substantiated index of As derived from beat-by-beat blood pressure (BP) response following an electrical stimulus. We have made physiological measurements including BP and found that the increased FIO2stimulus is effective in enhancing the AS. This finding was also confirmed in terms of the autonomic activity balance as well as the lengthening in time for active, safer, driving.

Physiological investigation of automobile driver's activation index using simulated monotonous driving.

Monotonous automobile operation in our daily life may cause the lowering of what might be termed an activation state of the human body, resulting in an increased risk of an accident. We therefore propose to create a more suitable environment in-car so as to allow active operation of the vehicle, hopefully thus avoiding potentially dangerous situations during driving. In order to develop such an activation method as a final goal, we have firstly focused on the acquisition of physiological variables, including cardiovascular parameters, during presentation to the driver of a monotonous screen image, simulating autonomous travel of constant-speed on a motorway. Subsequently, we investigated the derivation of a driver's activation index. During the screen image presentation, a momentary electrical stimulation of about 1 second duration was involuntarily applied to a subject's shoulder to obtain a physiological response. We have successfully monitored various physiological variables during the image presentation, and results suggest that a peculiar pattern in the beat-by-beat change of blood pressure in response to the involuntary stimulus may be an appropriate, and feasible, index relevant to activation state.

A new portable device for ambulatory monitoring of human posture and walking velocity using miniature accelerometers and gyroscope.

Measurement of physical activity is one of the key subjects in the field of ambulatory cardiovascular monitoring using such as Holter ECG and so called "ambulatory blood pressure monitor (ABPM)". Rehabilitation and gerontology will also be another fields for the application of the activity monitor. From this point of view, we have developed a portable device for monitoring human posture and walking velocity in ambulatory subjects. In this paper are described, a new sensor system for this purpose using three accelerometers and one gyroscope, its availability for the accurate measurement of human posture and walking velocity, and results of preliminary study using a prototype system for ambulatory monitoring.

Cloning and characterization of a chitosanase gene from the koji mold Aspergillus oryzae strain IAM 2660.

A genomic copy of the gene coding for chitosanase (csnA) was isolated from Aspergillus oryzae IAM 2660. A. oryzae csnA contains an open reading frame that encodes a polypeptide of 245 amino acids with a calculated molecular mass of 26,500 Da. The deduced amino acid sequence of A. oryzae csnA indicates extensive similarities to those of other fungal chitosanases.

L-Sorbose induces cellulase gene transcription in the cellulolytic fungus Trichoderma reesei.

L-Sorbose has previously been assumed to stimulate cellulase formation in an indirect manner, different from that of sophorose in Trichoderma reesei. Through Northern blot analysis however, L-sorbose was found to regulate coordinately six cellulase genes (including eg13, whose behavior has not been studied so far) at transcriptional level, as is the case with sophorose in T. reesei strains PC-3-7 and QM9414. Dot blot analysis showed that the proportions of each cellulase mRNA to cbh1 mRNA, the largest amount of mRNA transcribed in T. reesei, did not change when L-sorbose or sophorose was used as an inducer in the PC-3-7 and QM9414 strains. cbh2 and egl1 mRNAs were about 45-60% and 20-30% of the cbh1 transcript, whereas small amounts of mRNA, 1-2% of cbh1, were observed on other endoglucanase genes. Furthermore, the PC-3-7 strain showed an enhanced level of cellulase gene transcription, about two- and four- to six-fold higher than that of the QM9414 strain with sophorose and L-sorbose, respectively.

The bacterium Burkholderia gladioli strain CHB101 produces two different kinds of chitinases belonging to families 18 and 19 of the glycosyl hydrolases.

Two genes (chiA and chiB) coding for chitanases A and B (ChiA and ChiB) were isolated from the chitinolytic bacterium, Burkholderia gladioli strain CHB101. chiA contains an open reading frame that encodes a protein of 343 amino acids, whereas chiB encodes a protein of 307 amino acids. The deduced amino acid sequence of ChiA showed a high similarity to those of microbial chitinases belonging to family 18 of the glycosyl hydrolases, while ChiB showed significant sequence similarity to plant chitinases and Streptomyces spp. chitinases belonging to family 19.