Biomedical subjects

M Nogawa

Publications and source records attributed to M Nogawa.

At least 19 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2001

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.

Cellulase↗

PubMed2001

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.

Journal Article↗

PubMed2001

Role of cellulose-binding domain of exocellulase I from white rot basidiomycete Irpex lacteus.

The core fragment (designated P-42), devoid of the cellulose-binding domain (CBD) in the C-terminus and prepared from Irpex lacteus exocellulase I (Ex-1), was isolated by limited proteolysis using papain. Both the hydrolytic activity and binding ability of the isolated P-42 toward insoluble cellulose were lower than those of the native Ex-1, whereas Ex-1 and P-42 showed similar hydrolytic activities toward soluble substrates. These results indicate that the CBD of I. lacteus Ex-1 is the important domain which could enhance hydrolytic activity and binding ability of the enzyme toward insoluble cellulose. In addition, the isolated P-42 was different from the native Ex-1 in terms of enzymatic properties such as pH and temperature stabilities. These differences in stability, with regard to pH and temperature, between P-42 and the native Ex-1 are probably due to the O-linked sugar chains existing in the linker region.

Journal Article↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed2000

Regulation of xyn3 gene expression in Trichoderma reesei PC-3-7.

The characteristics of regulation of the gene encoding the third xylanase (Xyn III) of a filamentous fungus, Trichoderma reesei PC-3-7, were studied by Northern blot analysis. A partial DNA sequence (185 bp) of the xyn3 gene was obtained by PCR amplification of genomic DNA of T. reesei PC-3-7 and sequenced. This xyn3 gene fragment was used as a probe for Southern and Northern blot analysis. The expression of the xyn3 gene was regulated at the transcriptional level. The xyn3 mRNA was expressed in mycelia of T. reesei PC-3-7 induced by Avicel, L-sorbose and sophorose, but not by xylose, xylooligosaccharides and birchwood xylan. Furthermore, it was observed that xyn3 was synchronously expressed with egll but not with xyn1 and xyn2 by L-sorbose, indicating that the xyn3 gene may be coordinately expressed with cellulase genes. By Southern blot analysis, the xyn3 gene was confirmed to exist as a single copy in both strains of T. reesei PC-3-7 and QM9414. However, no xyn3 mRNA appeared in the mycelia induced by any kind of inducers in T. reesei QM9414 even when total RNA was used in large excess, suggesting that the xyn3 gene in T. reesei QM9414 is in the dormant state and cannot be expressed. Therefore, T. reesei PC-3-7 may be a very useful strain for elucidating the induction mechanism of xylanase biosynthesis by cellulosic and xylanosic substrates, and also the regulatory correlation between cellulase and xylanase induction.

Amino Acid Sequence↗

PubMed2000

Development of a compact, sealless, tripod supported, magnetically driven centrifugal blood pump.

In this study, a tripod supported sealless centrifugal blood pump was designed and fabricated for implantable application using a specially designed DC brushless motor. The tripod structure consists of 3 ceramic balls mounted at the bottom surface of the impeller moving in a polyethylene groove incorporated at the bottom pump casing. The follower magnet inside the impeller is coupled to the driver magnet of the motor outside the bottom pump casing, thus allowing the impeller to slide-rotate in the polyethylene groove as the motor turns. The pump driver has a weight of 230 g and a diameter of 60 mm. The acrylic pump housing has a weight of 220 g with the priming volume of 25 ml. At the pump rpm of 1,000 to 2,200, the generated head pressure ranged from 30 to 150 mm Hg with the maximum system efficiency being 12%. When the prototype pump was used in the pulsatile mock loop to assist the ventricle from its apex to the aorta, a strong correlation was obtained between the motor current and bypass flow waveforms. The waveform deformation index (WDI), defined as the ratio of the fundamental to the higher order harmonics of the motor current power spectral density, was computed to possibly detect the suction occurring inside the ventricle due to the prototype centrifugal pump. When the WDI was kept under the value of 0.20 by adjusting the motor rpm, it was successful in suppressing the suction due to the centrifugal pump in the ventricle. The prototype sealless, centrifugal pump together with the control method based on the motor current waveform analysis may offer an intermediate support of the failing left or right ventricle bridging to heart transplantation.

Centrifugation↗

PubMed1999

Expression of two Trichoderma reesei xylanases in the fission yeast Schizosaccharomyces pombe.

Two xylanase genes (xyn1 and xyn2) were amplified by the polymerase chain reaction (PCR) technique from first-strand cDNA prepared from mRNA of Trichoderma reesei QM9414. The genes were located under the human cytomegalovirus gene promoter (CMVp) on copy-number-controlled plasmids (pTLxyn1 and pTLxyn2). When both plasmids were introduced into Schizosaccharomyces pombe, functional xylanases (XYN I and XYN II) were secreted by the recombinant yeasts. The secreted XYN I protein had a molecular mass of 21 kDa whereas XYN II was produced as two molecular forms with sizes of 21 and 28 kDa, the former being not glycosylated and the latter N-glycosylated. XYN I was secreted in the culture medium at a level of about 25 microg/ml and XYN II at about 170 microg/ml. The recombinant xylanases had the same characteristics with respect to the effects of temperature and pH on the enzyme activity as the native ones.

Journal Article↗

PubMed1999

Ex vivo evaluation of a roller screw linear muscle actuator for an implantable ventricular assist device using trained and untrained latissimus dorsi muscles.

In this study, the power output and contraction length of trained and untrained canine latissimus dorsi (LD) muscles were measured using a roller screw linear muscle actuator (RSLMA). The RSLMA consisted of a roller screw-nut assembly and translation unit to convert the linear pull force of the muscle into an axial displacement of the roller screw. When a cable wound around a spool attached at each end of the roller screw nut was pulled in either direction, the nut was rotated which in turn advanced the roller screw in the axial direction. Under anesthesia, a Telectronics myostimulator (Model 7220) was implanted in the subcutaneous area of the canine left thoracic region with its bipolar intramuscular leads implanted around the thoracodorsal nerve and the distal muscle. A total of 6 dogs went through the myostimulator implantation, followed with 8 weeks of continuous stimulation. After completion of the training, the contraction lengths of the trained and untrained LD muscles were measured, and they were 4.25 and 5.5 cm, respectively, while the instantaneous power outputs were 4.24 and 8 W, respectively. Although untrained muscles could provide much higher instantaneous power immediately following the start of the stimulation, it diminished rapidly. On the other hand, the trained muscle showed prolonged fatigue resistance. The thoracolumbar and humeral approaches in attaching the actuator cable did not show a difference with respect to muscle power output. The trained LD muscle can provide sufficient power in the range of 3-4 W to drive a left heart assist device, but its long-term evaluation awaits development of an appropriate muscle-device interface for chronic in vivo application.

Animals↗

PubMed1999

Ultracompact, completely implantable permanent use electromechanical ventricular assist device and total artificial heart.

An ultracompact, completely implantable permanent use electromechanical ventricular assist device (VAD) and total artificial heart (TAH) intended for 50-60 kg size patients have been developed. The TAH and VAD share a miniature electromechanical actuator that comprises a DC brushless motor and a planetary roller screw. The rotational force of the motor is converted into the rectilinear force of the roller screw to actuate the blood pump. The TAH is a one piece design with left and right pusher plate type blood pumps sandwiching an electromechanical actuator. The VAD is one half of the TAH with the same actuator but a different pump housing and a backplate. The blood contacting surfaces, including those of the flexing diaphragm and pump housing, of both the VAD and TAH were made of biocompatible polyurethane. The diameter, thickness, volume, and weight of the VAD are 90 mm, 56 mm, 285 cc, and 380 g, respectively, while those of the TAH are 90 mm, 73 mm, 400 cc, and 440 g, respectively. The design stroke volume of both the VAD and TAH is 60 cc with the stroke length being 12 mm. The stroke length and motor speed are controlled solely based on the commutation signals of the motor. An in vitro study revealed that a maximum pump flow of 7.5 L/min can be obtained with a pump rate of 140 bpm against a mean afterload of 100 mm Hg. The power requirement ranged from 4 to 6 W to deliver a 4-5 L/min flow against a 100 mm Hg afterload with the electrical-to-hydraulic efficiency being 19-20%. Our VAD and TAH are the smallest of the currently available devices and suitable for bridge to transplant application as well as for permanent circulatory support of 50-60 kg size patients.

Evaluation Studies as Topic↗

PubMed1999

Measurement of blood hematocrit inside the magnetically suspended centrifugal pump using an optical technique: application to assessment of pump flow.

To measure blood hematocrit inside the magnetically suspended centrifugal pump, we have performed both forward and backward light scattering measurements using a specially designed optical cell. In the forward scattering measurement, an optical fiber was used to guide the near infrared light at 780 nm into a 250 microns gap region, and the light that forward scattered toward a detector fiber was measured using a phototransistor. The light intensity decreased exponentially with an increase in the hematocrit to around 20%. The forward scattering method suffered from sensitivity at the hematocrit levels around 25-45% due to the diffusion effect. By making the optical path length larger than several millimeters, the sensitivity of the forward scattering method in terms of hematocrit change can be improved. In the back scattering method, however, better sensitivity in terms of hematocrit change from 0-50% was obtained. By making the optical fiber separation distance less than 1 mm, the system will measure the first order back scattering from the shallow layer while, by making the fiber separation distance larger than several millimeters, the system will primarily measure the diffuse reflectance from the deeper layer. Both approaches will yield sensitive optical intensity change in terms of the physiological hematocrit range.

Algorithms↗

PubMed1999

Detection of suction and regurgitation of the implantable centrifugal pump based on the motor current waveform analysis and its application to optimization of pump flow.

In this study, a detection algorithm for suction and regurgitation of the centrifugal pump during left heart bypass without relying on external flow or pressure sensors was developed and evaluated in acute studies using adult goats. The detection scheme relies on power spectral density (PSD) analysis of the motor current waveform through which the waveform deformation index (WDI) is obtained. This index is defined as the ratio of the fundamental component of the PSD to the higher PSD components, and its value increases with the deformation of the basic waveform. By assuming that the undistorted motor current waveform can be represented by a pure sine waveform, we theoretically synthesized various waveforms which have different second harmonic components. We were able to synthesize the waveform whose shape was close to the distorted motor current waveform under varying suction levels obtained in a mock loop study. From this study, we came to the conclusion that the WDI value of 0.2 can serve as a threshold level in deciding the suction and regurgitation speeds (rpm) during left heart bypass. In the study using adult goats, we were successful in minimizing both regurgitation and suction when the centrifugal pump speed was adjusted based on the WDI algorithm. The resultant bypass flow ranged from 1.5 to 2.0 L/min which was around 60% of the total flow. Further study is underway to evaluate the applicability of the WDI method in optimizing bypass pump flow.

Algorithms↗

PubMed1999

An alpha-L-arabinofuranosidase from Trichoderma reesei containing a noncatalytic xylan-binding domain.

L-Sorbose, an excellent cellulase and xylanase inducer from Trichoderma reesei PC-3-7, also induced alpha-L-arabinofuranosidase (alpha-AF) activity. An alpha-AF induced by L-sorbose was purified to homogeneity, and its molecular mass was revealed to be 35 kDa (AF35), which was not consistent with that of the previously reported alpha-AF. Another species, with a molecular mass of 53 kDa (AF53), which is identical to that of the reported alpha-AF, was obtained by a different purification procedure. Acid treatment of the ammonium sulfate-precipitated fraction at pH 3.0 in the purification steps or pepsin treatment of the purified AF53 reduced the molecular mass to 35 kDa. Both purified enzymes have the same enzymological properties, such as pH and temperature effects on activity and kinetic parameters for p-nitrophenyl-alpha-L-arabinofuranoside (pNPA). Moreover, the N-terminal amino acid sequences of these enzymes were identical with that of the reported alpha-AF. Therefore, it is obvious that AF35 results from the proteolytic cleavage of the C-terminal region of AF53. Although AF35 and AF53 showed the same catalytic constant with pNPA, the former showed drastically reduced specific activity against oat spelt xylan compared to the latter. Furthermore, AF53 was bound to xylan rather than to crystalline cellulose (Avicel), but AF35 could not be bound to any of the glycans. These results suggest that AF53 is a modular glycanase, which consists of an N-terminal catalytic domain and a C-terminal noncatalytic xylan-binding domain.

Amino Acid Sequence↗

PubMed1998

Purification and Characterization of Exo-beta-d-Glucosaminidase from a Cellulolytic Fungus, Trichoderma reesei PC-3-7.

Chitosan-degrading activities induced by glucosamine (GlcN) or N-acetylglucosamine (GlcNAc) were found in a culture filtrate of Trichoderma reesei PC-3-7. One of the chitosan-degrading enzymes was purified to homogeneity by precipitation with ammonium sulfate followed by anion-exchange and hydrophobic-interaction chromatographies. The enzyme was monomeric, and its molecular mass was 93 kDa. The optimum pH and temperature of the enzyme were 4.0 and 50 degrees C, respectively. The activity was stable in the pH range 6.0 to 9.0 and at a temperature below 50 degrees C. Reaction product analysis from the viscosimetric assay and thin-layer chromatography and H nuclear magnetic resonance spectroscopy clearly indicated that the enzyme was an exo-type chitosanase, exo-beta-d-glucosaminidase, that releases GlcN from the nonreducing end of the chitosan chain. H nuclear magnetic resonance spectroscopy also showed that the exo-beta-d-glucosaminidase produced a beta-form of GlcN, demonstrating that the enzyme is a retaining glycanase. Time-dependent liberation of the reducing sugar from partially acetylated chitosan with exo-beta-d-glucosaminidase and the partially purified exo-beta-d-N-acetylglucosaminidase from T. reesei PC-3-7 suggested that the exo-beta-d-glucosaminidase cleaves the glycosidic link of either GlcN-beta(1-->4)-GlcN or GlcN-beta(1-->4)-GlcNAc.

Journal Article↗

PubMed1997

Neurocrescin: a novel neurite-outgrowth factor secreted by muscle after denervation.

Regeneration of injured axons at neuromuscular junctions has been assumed to be regulated by extracellular factors that promote neurite outgrowth. We report here the cloning of a novel neurite outgrowth factor, designated neurocrescin, from chick denervated skeletal muscle. A recombinant neurocrescin promoted neurite outgrowth from cultured neurons of spinal cord and telencephalon of chick embryo. It was expressed predominantly in neural tissue and muscle, and was secreted extracellularly after intramolecular cleavage. This truncated form was detected in denervated muscle but not in innervated muscle. Thus, neurocrescin appears to be a novel neurite outgrowth factor that is secreted in an activity-dependent fashion. A highly homologous counterpart was also cloned from mouse brain.

Amino Acid Sequence↗

PubMed1997

Demonstration of functionally distinct human polymorphonuclear leukocyte fractions by simultaneous measurement of phagocytosis and oxygen radical generation.

Phagocytosis and oxygen radical generation by human polymorphonuclear leukocytes (PMNLs) were studied by a two-color flow cytometric analysis, where the red fluorescent product(s) of hydroethidine was used as an indicator of intracellular generation of oxygen radicals and opsonized zymosan (OZ) as an indicator of phagocytosis. Unstimulated cells formed a single population of cells without any significant fluorescence. PMNLs stimulated by OZ exhibited a high red fluorescence. Most PMNLs phagocytosed OZ and generated oxygen radicals when stimulated by zymosan particles opsonized with human AB serum at concentrations of more than 10%, whereas three distinct subpopulations (designated as R1, R2 and R3) appeared when stimulated by zymosan particles opsonized with 3% serum; R1 cells enhanced neither green nor red fluorescence, R2 cells enhanced green fluorescence but not red fluorescence, and R3 cells enhanced both green and red fluorescence. The R2 cells completely disappeared by the addition of Trypan blue. Most of the R3 cells disappeared by the addition of cytochalasin B. These findings on the three fractions were confirmed by the observation under fluorescence microscopy of cells in each fraction obtained by sorting. In conclusion, PMNLs could be separated into three functionally distinct subpopulations when stimulated by zymosan particles opsonized with a suboptimal concentration of serum.

Cell Differentiation↗

PubMed1997

Control of centrifugal blood pump based on the motor current.

In this study, centrifugal pump performance was examined in a mock circulatory loop to derive an automatic pump rotational speed (rpm) control method. The pivot bearing supported sealless centrifugal pump was placed in the left ventricular apex to aorta bypass mode. The pneumatic pulsatile ventricle was used to simulate the natural ventricle. To simulate the suction effect in the ventricle, a collapsible rubber tube was placed in the inflow port of the centrifugal pump in series with the apex of the simulated ventricle. Experimentally, the centrifugal pump speed (rpm) was gradually increased to simulate the suction effect. The pump flow through the centrifugal pump measured by an electromagnetic flowmeter, the aortic pressure, and the motor current were continuously digitized at 100 Hz and stored in a personal computer. The analysis of the cross-spectral density between the pump flow and motor current waveforms revealed that 2 waveforms were highly correlated at the frequency range between 0 and 4 Hz, with the coherence and phase angles being close to 1.0 and 0 degree, respectively. The fast Fourier transform analysis of the motor current indicated that the second harmonic component of the motor current power density increased with the occurrence of the suction effect in the circuit. The ratio of the fundamental to the second harmonic component decreased less than 1.3 as the suction effect developed in the circuit. It is possible to detect and prevent the suction effect of the centrifugal blood pump in the natural ventricle through analysis of the motor current waveform.

Algorithms↗