Search

Mammalian glycerophosphodiesterases (GDEs) were recently shown to be involved in multiple cellular signaling pathways. This study showed that decreased GDE5 expression results in accumulation of intracellular glycerophosphocholine (GPC), showing that GDE5 is actively involved in GPC/choline metabolism in 3T3-L1 adipocytes. Using 3T3-L1 adipocytes, we further studied the biological significance of GPC/choline metabolism during adipocyte differentiation. Inhibition of GDE5 suppressed the formation of lipid droplets, which is accompanied by the decreased expression of adipocyte differentiation markers. We further showed that the decreased GDE5 expression suppressed mitotic clonal expansion (MCE) of preadipocytes. Decreased expression of CTP: phosphocholine cytidylyltransferase (CCTβ), a rate-limiting enzyme for phosphatidylcholine (PC) synthesis, is similarly able to inhibit MCE and PC synthesis; however, the decreased GDE5 expression resulted in accumulation of intracellular GPC but did not affect PC synthesis. Furthermore, we showed that mRNAs of proteoglycans and transporters for organic osmolytes are significantly upregulated and that intracellular amino acids and urea levels are altered in response to GDE5 inhibition. Finally, we showed that reduction of GDE5 expression increased lactate dehydrogenase release from preadipocytes. These observations indicate that decreased GDE5 expression can suppress adipocyte differentiation not through the PC pathway but possibly by intracellular GPC accumulation. These results provide insight into the roles of mammalian GDEs and their dependence upon osmotic regulation by altering intracellular GPC levels.

To optimize the preparation methods for liposomes encapsulating mercaptoundecahydrododecaborate (BSH), we examined BSH and lipid concentrations that increased the boron content in liposomes. We improved the BSH encapsulation efficiency and boron content of the liposomes from 4.2 to 45.9 % and 9.5–54.3 μg, respectively, by changing the lipid concentration from 10 to 150 mg/mL. Notably, the boron content increased significantly from 26.2 μg to 326.3 μg at a constant lipid concentration of 30 mg/mL with increased BSH concentrations.

Background Appropriate hemostasis is essential for clear visualization of the neural structures and cleavage planes. It is also essential for avoiding heat-induced injury, minimizing blood loss, and reducing operative time. Objective To determine the role of cerebrospinal fluid (CSF) in platelet-dependent hemostasis during neurosurgery. Methods The amplification of aggregation, activation of integrin αIIbβ3, intrinsic and extrinsic coagulation pathways, and activation of signaling cascades in platelets were evaluated. For comparison, various concentrations of a commercially available artificial CSF solution (aCSF), an artificial CSF solution prepared by the authors, and normal saline (NS) were used. Differences between aCSF and NS in obtaining in vivo hemostasis were assessed by measuring the tail vein bleeding time in C57BL/6N mice. Results Platelet aggregation was directly amplified by the addition of aCSF through increased activation of integrin αIIbβ3, phosphatidylserine exposure, and P-selectin expression. However, the prothrombin time and activated partial thromboplastin time were not primarily related to coagulation activity with the addition of aCSF. Activation of Src kinase was related to platelet activation by aCSF. The elimination of sodium bicarbonate from aCSF and the addition of the selective inhibitor of the HCO3/Cl exchanger, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt, significantly inhibited platelet aggregation. The bleeding time in aCSF-treated mice was significantly shorter than that in NS-treated mice. Conclusion Sodium bicarbonate facilitates hemostasis through the amplification of platelet aggregation function. The existence of CSF and irrigation with aCSF provide better conditions for physiological hemostasis and they have the potential of improving hemostasis by bipolar coagulation or with irrigation during neuroendoscopic procedures.

Particle-induced gamma-ray emission (PIGE), which measures prompt gamma rays at 428 keV from 10B (p, p'γ) 7Be, was used to confirm the boron distribution within 2hr-BPA-exposed cells. Distribution images of potassium, phosphate, and boron and the whole spectrum showed the ratios of boron counts to total (%) as follows: control group: 1.35 ± 0.073%; 2hr boron exposure group: 2.33 ± 0.35%; and boron exposure/wash group: 1.58 ± 0.095%. Micro-beam PIXE/PIGE can be a promising tool for visualization of intracellular Boron.

Tumor sphere-forming (TS) glioma stem cells and cancerous TS cells were analyzed in vivo and in vitro. The boron concentration in murine TS tumors was higher than normal tissue. The boron concentration at 24 h was 0.80 ± 0.09 μg/107 in the TS cells, and 1.08 ± 0.08 μg/107 in the cancerous cells. The LAT-1 amino-acid transporter positive rate was 35.4% in the TS cells and 100% in the cancerous cells. These results suggested the relation between LAT-1 expression and boronophenylalanine concentration in vitro.

The neutron source of BNCT is currently changing from reactor to accelerator, but peripheral facilities such as a dose-planning system and blood boron analysis have still not been established. To evaluate the potential application of particle-induced gamma-ray emission (PIGE) for boron measurement in clinical boron neutron capture therapy, boronophenylalanine dissolved within a cell culture medium was measured using PIGE. PIGE detected 18 μgB/mL f-BPA in the culture medium, and all measurements of any given sample were taken within 20 min. Two hours of f-BPA exposure was required to create a boron distribution image. However, even though boron remained in the cells, the boron on the cell membrane could not be distinguished from the boron in the cytoplasm.

application/pdf, Objective We aimed to clarify the optimal timing for the fluorescence imaging of brain tumor tissue differentiated from brain edema after the administration of photosensitizers. Methods We have performed an in vivo study of the kinetics of 5-aminolevulinic acid (5-ALA) in comparison with talaporfin sodium using the rat brain tumor model and rat vasogenic edema model produced by cold injury. The in vivo kinetics of 5-ALA and talaporfin sodium in brain tumor model and the vasogenic edema model was determined by a fluorescence macroscope and a microplate reader. Results The in vivo kinetic study of 5-ALA showed mild fluorescence intensity of protoporphyrin IX (PpIX) in brain tumor differentiated from vasogenic edema. The mean lesion-to-normal-brain ratio (L/N ratio) in the group of brain tumor model 2 h after the administration of 5-ALA was 7.78 ± 4.61, which was significantly higher (P < 0.01) than that of the vasogenic edema 2 h after the administration of 5-ALA (2.75 ± 1.12). In vivo kinetic study of talaporfin sodium showed high fluorescence intensity and retention in brain tumor differentiated from vasogenic edema. The mean L/N ratio of the fluorescence intensity in the group of brain tumor model 12 h after the administration of talaporfin sodium was 23.1 ± 11.9, which was significantly higher (P < 0.01) than that of the vasogenic edema 12 h after the administration (8.93 ± 8.03). Conclusions The optimization of fluorescence imaging of brain tumors differentiated from brain edema is possible in the case of 5-ALA within 6 h, and also possible in the case of talaporfin sodium beyond 12 h.

A simple new method for making a rat post-angioplasty stenosis model was developed using a single-lumen compliant balloon catheter/guidewire system and no special diet. This technique was applied to 10 9-week-old Wistar rats fed a normal diet. The catheter/guidewire system was inserted from the external carotid artery and advanced retrogradely into the common carotid artery. The balloon was inflated six times with rotation through 60°. After angioplasty, the external carotid artery was ligated. Homogeneous stenoses due to intimal hyperplasia were demonstrated in cross sections of all cases. This simple and gentle method allows an easily reproducible post-angioplasty stenosis model.

Hyaluronic acid (HA), a nonimmunogenic, biocompatible polymer found in different biological tissues, has the potential to attach to CD44 receptors on the surface of certain cancer cells, where the receptor is overexpressed compared with normal cells. Boron-hyaluronic acid (BHA) was tested for its feasibility as a potential agent for BNCT. BHA with low-viscosity 30 kDa HA could be administered by intravenous injection. The compound showed a certain degree of cytotoxicity and accumulation in C6 rat glioma cells in vitro. Instability of the chelate bonds between boron and HA and/or insufficient specificity of CD44 receptors on C6 cells to BHA could account for the insufficient in vitro accumulation. To ensure the future eligibility of BHA for BNCT experiments, using alternative tumor cell lines and chemically securing the chelate bonds or synthesizing BHA with boron covalently attached to HA might be required.

Because of its fast metabolism gadolinium as a commercial drug was not considered to be suitable for neutron capture therapy. We studied additive effect of gadolinium and boron co-administration using colony forming assay. As a result, the survival of tumor cells with additional 5 ppm of Gd-DTPA decreased to 1/10 compared to the cells with boron only. Using gadolinium to increase the effect of BNCT instead of additional X-ray irradiation might be beneficial, as such combination complies with the short-time irradiation regimen at the accelerator-based neutron source.

We evaluated DNA double-strand breaks (DSBs) induced by boron neutron capture reaction (BNCR) using plasmid DNA, boron solution, and gel electrophoresis. The amount of the linear form of DNA produced by DSBs increased with the neutron-beam irradiation dose. The amount of the open-circular form of DNA produced by single-strand breaks (SSBs) increased with the neutron-beam irradiation dose and the (10)B concentration. The model facilitated quantification of BNCR-induced DSBs and SSBs, irrespective of the DNA repair mechanism.

In neutron capture therapy, it is important that the boron is selectively uptaken by tumor cells. In the present study, we used flow cytometry to sort the cells in the G0/G1 phase and those in the G2/M phase, and the boron concentration in each fraction was measured with inductively coupled plasma atomic emission spectroscopy. The results revealed that sodium borocaptate and boronophenylalanine (BPA), were associated with higher rates of boron uptake in the G2/M than in the G0/G1 phase. However, the difference was more prominent in the case of BPA. The G2/M:G0/G1 ratio decreased as a function of exposure time in BPA containing culture medium, thereby indicating the cell cycle dependency of BPA uptake. Such heterogeneity of boron uptake by tumor cells should be considered for microdosimetry.

A new boronated porphyrin compound (STA-BX909) was developed as a possible agent for boron neutron capture therapy. The boron concentration was measured by an in vivo rat experimental brain tumor model and an in vitro cell culture study. This agent was compared to sodium borocaptate (BSH) which has been used in clinical trials of boron neutron capture therapy. In the 9L rat brain tumor model, STA-BX909 achieved a higher boron tumor/blood ratio 24 h after injection in comparison to BSH. A boron concentration study in cultured glioma cell lines (U-251, U-87, 9L) demonstrated an increased boron concentration as a function of exposure time to STA-BX909, while the boron concentration remained stable with increasing exposure time to BSH. Use of a colony forming assay with thermal neutron irradiation revealed more cytotoxicity with STA-BX909 than BSH when the same concentration of 10B was administered. We concluded that STA-BX909 may be an effective drug for use in boron neutron capture therapy and that it merits further investigation.

We used a two-color analysis system to assess the porphyrin uptake and DNA content in four established cell lines of glioma employing flow cytometry (FCM). The FCM study revealed porphyrin uptake in all cells, regardless of the phase of the cell cycle they were in. However, those in the G0/G1 phase showed moderate uptake of porphyrin and those in the G2/M phase showed a higher uptake. These results indicated the advantage of using porphyrin as the carrier of tumor targeting agents as a therapeutic strategy for malignant tumors.

BPA used in BNCT has a similar structure to some essential amino acids and is transported into tumor cells by amino acid transport systems. Previous study groups have tried various techniques of loading BPA to increase intracellular boron concentration. CHO-K1 cells demonstrate system L (LAT1) activity and are suitable for specifying the transport system of a neutral amino acid. In this study, we examined the intracellular accumulation of boron in CHO-K1 cells by amino acid transport control, which involves co-loading with L-type amino acid esters. Intracellular boron accumulation in CHO-K1 cells showed the greatest increased upon co-loading 1.0 mM BPA, with 1.0 mM l-Tyr-O-Et and incubating for 60 min. This increase is caused by activation of a system L amino acid exchanger between BPA and l-Tyr. The amino acid esters are metabolized to amino acids by intracellular hydrolytic enzymes that increase the concentrations of intracellular amino acids and stimulate exchange transportation. We expect that this amino acid transport control will be useful for enhancing intracellular boron accumulation.

We have evaluated the efficacy and safety of boron neutron capture therapy (BNCT) for recurrent glioma and malignant brain tumor using a new protocol. One of the two patients enrolled in this trial is a man with recurrent glioblastoma and the other is a woman with anaplastic meningioma. Both are still alive and no severe adverse events have been observed. Our findings suggest that NCT will be safe as a palliative therapy for malignant brain tumors.

The phase II trial has been prepared to assess the effectiveness of BPA (250 mg/kg)-based NCT combined with X-ray irradiation and temozolomide (75 mg/m2) for the treatment of newly diagnosed GBM. BPA uptake is determined by 18F-BPA-PET and/or 11C-MET-PET, and a tumor with the lesion to normal ratio of 2 or more is indicated for BNCT. The maximum normal brain point dose prescribed was limited to 13.0 Gy or less. Primary end point is overall survival.

We measured the toxicity and intracellular uptake of a newly developed boronated porphyrin EC032, and verified the fluorescence-based boron concentration measuring methods. Toxicity study showed that concentration required to produce a 50% reduction in viability (IC(50)) of EC032 was more than 0.25 mM. Fluorescence study showed the intracellular uptake of EC032 increased up until 24 h after its exposure to C6, 9L, U87, and U251 cells. There was also a linear correlation between ICP-AES and fluorescence intensity as an arbitrary unit about measurement of boron concentration. Fluorescence-based boron concentration measuring methods are very simple and useful methods, especially for screening of slight test dose of porphyrin compounds.

A simple new method for making a rat post-angioplasty stenosis model was developed using a single-lumen compliant balloon catheter/guidewire system and no special diet. This technique was applied to 10 9-week-old Wistar rats fed a normal diet. The catheter/guidewire system was inserted from the external carotid artery and advanced retrogradely into the common carotid artery. The balloon was inflated six times with rotation through 60 degrees . After angioplasty, the external carotid artery was ligated. Homogeneous stenoses due to intimal hyperplasia were demonstrated in cross sections of all cases. This simple and gentle method allows an easily reproducible post-angioplasty stenosis model.

We have previously reported that buthionine sulfoximine (BSO) enhances sodium borocaptate (BSH) uptake by down regulating glutathione (GSH) synthesis in cultured cells. This study investigated the influence of BSO on tissue BSH uptake in vivo and the efficacy of BSH–BSO-mediated boron neutron capture therapy (BNCT) on tumor growth using a Fisher-344 rat subcutaneous tumor model. With BSO supplementation, boron uptake in subcutaneous tumor, blood, skin, muscle, liver, and kidney was significantly enhanced and maintained for 12 h. Tumor growth was significantly delayed by using BSO. With further improvement in experimental conditions, radiation exposure time, together with radiation damage to normal tissues, could be reduced.

Sodium borocaptate (BSH) is widely used for boron neutron capture therapy (BNCT) of brain tumors. However, the mechanism of uptake by the tumor remains unclear. We investigated the sulfhydryl moiety of this compound. Down regulation of glutathione (GSH) by buthionine sulfoximine in cultured cells resulted in increase of BSH uptake (7.9-36.5%) compared to the control group and consequently the cytocidal effect of neutron irradiation also increased. On the other hand, the radiation caused damage by gamma-ray irradiation was suppressed when BSH uptake increased. These findings suggested that modulation of GSH enhanced the effect of B (n, alpha) reaction and the protective effect of secondary gamma-ray in BNCT.

application/pdf, We examined the simultaneous uptake of porphyrin and (LDL) by four established cell lines of glioma and normal ®broblasts using ¯ow cytometry (FCM). The results indicated porphyrin and LDL showed competitive conjugation with the LDL receptor. These results support the theory of the porphyrin uptake via the LDL receptor.

We examined the incidence of radiation-induced apoptosis in 9L gliosarcoma cells in response to gamma-ray irradiation (single doses of 1, 10, or 20Gy), neutron irradiation, and BNCT using BSH or boronated metalloporphyrin (STA-BX9O9). In control (unirradiated) cells, the incidence of apoptosis was below 1%. In the gamma-ray group, no significant increase of apoptotic cells was observed at 1 Gy. In the neutron and the 20Gy gamma-ray groups, a significant increase (5.5%) of apoptosis was noted. There were few detectable apoptotic cells in the BNCT groups. Differences were seen not only in the cell-killing efficacy but also in the mode of cell death among in vitro gamma irradiation, neutron irradiation, and BNCT. Apoptosis is a possible biological marker of heterogeneous response of tumor cells following BNCT.

Porphyrin is known as a tumor localizing agent that is clinically used in photo-dynamic therapy for cancers.1 Boronated porphyrins have also been developed2, 3, 4, 5, 6, 7 and are now being investigated for possible clinical use.2

To improve boron neutron capture therapy (BNCT), a new tumor-seeking drug is required. Boronated porphyrins have been developed for boron carriers in boron neutron capture therapy (BNCT) with some positive results.1, 2, 3, 4, 5 We have developed a boronated metalloporphyrin compound, STA-BX900, and previously reported its significance compared to borocaptate sodium (BSH).3 To improve the delivery of such compounds to the tumor, two other types of boron porphyrin compounds, whose side chains were slightly modified, have been evaluated. The in vivo and in vitro uptake study of these drugs will be presented.

Proliferating tumor cell progress through the phases of each cell cycle. Cell cycle change such as G2/M arrest is known to occur in tumor cells after γ ray radiation.1,2 However, cell cycle change of tumor cell after neutron capture therapy has not been reported. We irradiated thermal neutron to 9L gliosarcoma cell in vitro with or without boron compounds and examined cell cycle changes of tumor cells with flow cytometry.

The potential for using boronated porphyrins for BNCT have been reported by some groups.1, 2, 3, 4, 5 Our recent studies have also shown that porphyrin is one of the potential tumor targeting agents for BNCT.6,7 The tumor specific uptake of porphyrin is well known, however the uptake of porphyrin regarding the cell cycle has not been reported. We examined tumor cell uptake of porphyrin in the relation with cell cycle using flow cytometry system in in vitro glioma cell lines.

We examined whether selective tumor accumulation of a novel manganese-metalloporphyrin (ATN-10) occurs in Fisher rats bearing intracerebral 9L gliomas.After intravenous administration of ATN-10, magnetic resonance imaging of brains with tumors or nontumoral vasogenic brain edema was performed. Tissue manganese concentrations were measured by inductively coupled plasma atomic emission spectroscopy until 48 hours after administration of ATN-10, to evaluate its uptake in tumor, normal brain, and peritumoral brain tissue.In magnetic resonance imaging scans, early enhancement was observed in both tumor tissue and regions of nontumoral vasogenic brain edema at 5 minutes after ATN-10 administration. However, delayed enhancement was noted only in tumor tissue, at 24 hours after intravenous injection of ATN-10. Comparison of rat brain specimens and 24-hour magnetic resonance imaging scans revealed that only the viable portions of tumors were enhanced with ATN-10; necrotic regions and areas of peritumoral brain tissue and nontumoral vasogenic edema were not. Significantly greater uptake of ATN-10 was found in tumor samples, compared with normal and peritumoral brain tissue, at 24 hours. A high tumor/normal brain tissue ratio (10.4) was achieved at 24 hours.ATN-10, a manganese-metalloporphyrin, is a potentially useful tumor-localizing agent that accumulates and is preferentially retained in viable tumor tissue.

The authors regret that in the printed version of the above-mentioned paper a number of errors occurred. These errors are now corrected below. On page 135, the first sentence in the second paragraph should read as follows: In boron neutron capture therapy (BNCT), due to the short range of alpha particles (about 10 mm), only the cells that incorporate the boron compounds are selectively injured. On page 138, the sentences from the second line should read as follows: The protein content of the cells in the G0/G1 phase was 2.06 ^ 0.24 mg in SCCVII, 1.47 ^ 0.09 mg in C6 and 1.40 ^ 0.01 mg in V79. Each value corresponds to 10 cells. In the G2/M phase it was 3.23 ^ 0.13, 2.82 ^ 0.04 and 2.62 ^ 0.12 mg, respectively. The protein content in cells in the G2/M phase was 1.57– 1.92 times higher than that in cells in the G0/G1 phase. The boron concentration was re-calculated based on the protein content of cells in each cell cycle phase. As a result, the G0/G1: G2/M ratios in the BSH group was 1.37 for SCCVII, 0.94 foe C6 and 0.55 for V79. In the BPA group, the ratio was 1.40 for SCCVII, 1.62 for C6, and 0.84 foe V79 (Fig. 5). On page 139, the second sentence starting from second line should read as follows: BSH is known to accumulate in tumor cells only and is scarcely measurable in the normal brain, but blood boron concentration to tumor boron concentration ratio is reported to be around one depending on the tumor sample [5, 6]. On page 140, the sentence starting from 9th line in the right column should read as follows: The G0/G1: G2/M ratio in BPA calculated by either cell number or protein content (1.40 in SCCVII, 1.62 in C6 tumor cell lines) should be considered critically because of the short path length of alpha particles.

Transglucosylation of an α-glucosidase (I) from honeybee was investigated. The honeybee α- glucosidase catalyzed the predominant formation of α-1,4-glucosidic linkage from sucrose, phenyl α-glucoside, or maltose. The major product from sucrose was 4G-a-glucosyl-sucrose (erlose), which differs from the report by Huber and Thompson that 3F-a-glucosyl-sucrose (melezitose) is mainly synthesized from sucrose by the transglucosylation of the same α-glucosidase: Biochemistry, 12, m 4011 (1973). Melezitose was not detected as the transglucosylation product of this enzyme.