Synthesis and biological evaluation of new boron-containing chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer.

New boron-containing chlorin derivatives 9 and 13 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized from photoprotoporphyrin IX dimethyl ester (2) and L-4-boronophenylalanine-related compounds. The in vivo biodistribution and clearance of 9 and 13 were investigated in tumor-bearing mice. The time to maximum accumulation of compound 13 in tumor tissue was one-fourth of that of compound 9, and compound 13 showed rapid clearance from normal tissues within 24h after injection. The in vivo therapeutic efficacy of PDT using 13 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3h after injection of 13. Tumor growth was significantly inhibited by PDT using 13. These results suggested that 13 might be a good candidate for both PDT and BNCT of cancer.

Isolation of new indole alkaloid triglucoside from the aqueous extract of Uncaria rhynchophylla.

Uncaria rhynchophylla (Miq.) Miq. (Rubiaceae) is widely used as a botanical raw material for traditional Japanese and Chinese medicines. However, not all of its potentially bioactive constituents have been isolated and characterized. Herein, one new indole alkaloid triglucoside (1), nine known alkaloids (2-10) and thirteen known non-alkaloids (11-23) were isolated from the aqueous extract of Uncaria rhynchophylla hook and structurally characterized 1H and 13C NMR and high-resolution electrospray ionization mass spectrometry. The absolute configurations of isolated compounds (1, 2 and 3) were determined by the X-ray diffraction analysis of their single crystals obtained using a micro-drop crystallization technique. This technique allows single crystals to be obtained from samples as small as 50 µg, thus providing detailed structural information even on minor constituents and enabling the accurate quality monitoring of botanical raw materials more accurately.

Synthesis and biological evaluation of new chlorin derivatives as potential photosensitizers for photodynamic therapy.

Three new water-soluble chlorin derivatives 3, 5 and 8 for potential use as photosensitizers in photodynamic therapy (PDT) for cancer were synthesized from photoprotoporphyrin IX dimethyl ester (1). The in vivo biodistribution and clearance of chlorin derivatives 3, 5 and 8 were investigated in tumor-bearing mice. Iminodiacetic acid derivative 8 showed the greatest tumor-selective accumulation among the new chlorin derivatives with maximum accumulation in tumor tissue at 3h after intravenous injection and rapid clearance from normal tissues within 24h after injection. The in vivo therapeutic efficacy of PDT using 8 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3h after injection of 8. Tumor growth was significantly inhibited by PDT using 8. These results indicate that iminodiacetic acid derivative 8 is useful as a new photosensitizer to overcome the disadvantages of photosensitizers that are currently in clinical use.

[Quantitative Determination Method of Aconitum Monoester Alkaloids Using Relative Molar Sensitivity (RMS) for the Assay in the Japanese Pharmacopoeia].

Recently, a novel quantitative method using relative molar sensitivity (RMS) was applied to quantify the ingredients of drugs and foods. An important development in this regard can be observed in the Japanese Pharmacopoeia (JP) 18, where the quantification of perillaldehyde, an unstable compound, in crude drug "Perilla Herb," was revised to incorporate the RMS method. In this study, the primary objective was to improve the tester safety and reduce the amount of reagents used in the JP test. To achieve this, the quantification of three toxic Aconitum monoester alkaloids (AMAs) was explored using the RMS method, employing a single reference compound for all three targets. These AMAs, namely benzoylmesaconine hydrochloride, benzoylhypaconine hydrochloride, and 14-anisoylaconine hydrochloride, which are the quantitative compounds of Kampo extracts containing Aconite Root (AR), were quantified using the reference compound benzoic acid (BA). Reliable RMS values were obtained using both 1H-quantitative NMR and HPLC/UV. Using the RMS of three AMAs relative to the BA, the AMA content (%) in commercial AMAs quantitative reagents were determined without analytical standards. Moreover, the quantitative values of AMAs using the RMS method and the calibration curve method using the three analytical standards were similar. Additionally, similar values were achieved for the three AMAs in the Kampo extracts containing AR using the RMS and the modified JP18 calibration curve methods. These results suggest that the RMS method is suitable for quantitative assays of the Kampo extracts containing AR and can serve as an alternative to the current method specified in the JP18.

Trace and major elements status in bronchoalveolar lavage fluid in dogs with or without bronchopneumonia.

The aim of this study was to investigate the relationships between the bronchopneumonia and mean concentrations of those trace elements in bronchoalveolar lavage fluid (BALF). Twenty-nine dogs were included this study (17 healthy dogs and 12 dogs with respiratory disease). Each BALF sample had been obtained during bronchoscope examination by use of a standardized method. The concentrations of Al, Br, Ca, Cu, Fe, K, Ni, P, Si, Sr and Zn in BALF were measured by the particle-induced X-ray emission method. We found no relationship between the bronchopneumonia and the levels of elements in the BALF, except Ca, P and Zn. The dogs with respiratory disease were found to have a large amount of Ca and Zn, and a high Ca/P and Zn/Cu ratios in BALF compared to those without respiratory disease.

The effect of 7.2% hypertonic saline solution with 6% dextran 70 on cardiac contractility as observed by an echocardiography in normovolemic and anesthetized dogs.

We studied the effect of a small volume of 7.2% hypertonic saline solution (HSS) or HSS with 6% dextran 70 (HSD) on hemodynamic status, especially on cardiac contractility, in anesthetized dogs using the left ventricular end-systolic volume index (ESVI) and ejection fraction (EF), which can be obtained in noninvasive echocardiography. In the present study, the mean values of ESVI were unaffected by HSS and HSD infusion, whereas the left ventricular end-diastolic volume index (EDVI) was markedly and significant increased. As a result of the changes in EDVI but not in ESVI, EF increased transiently and significantly in the HSS and HSD group, whereas no such significant change was observed in the dogs that received isotonic saline solution. In addition, as a result of the increases in cardiac index but not arterial pressure, system vascular resistances (SVR) decreased transiently and significantly in the HSS and HSD groups, whereas no such significant change was observed in the ISS group. Therefore, the positive inotropic effects of HSS and HSD may be attributable to the increase in left ventricular preload and decreases in SVR rather than direct changes in myocardial contractility.

Negative growth control of osteosarcoma cell by Bowman-Birk protease inhibitor from soybean; involvement of connexin 43.

Bowman-Birk protease inhibitor (BBI) from soybean acts as a potential chemopreventive agent in several types of tumors. However, the mechanism is still unclear. The present study was undertaken to estimate a mechanism of BBI-dependent negative growth control of human osteosarcoma cell (U2OS cell). BBI had negative growth control of the cells via induction of connexin (Cx) 43, a tumor suppressor gene in U2OS cells. This negative growth control by BBI was abrogated under down-regulation of Cx43 induced by a Cx43 antisense nucleotide treatment. It was also found that the BBI-dependent induction of Cx43 was due to elevation of Cx43 mRNA and stabilization of Cx43 protein. Especially, BBI-dependent inhibition of chymotrypsin-like activity in proteasome contributed to stabilization of Cx43 protein. These results suggest that a major negative growth effect of BBI is based on the restoration of Cx43 expression in U2OS cells.

Relationship between hair elements and severity of atrioventricular block in horses.

The aim of this study was to investigate the relationships between the mean concentrations of trace elements and the severity of the second-degree atrioventricular (AV) block in the mane hair of horses. Electrocardiographs of horses were continually recorded for 6 h using a holter cardiac monitor to determine dropped ventricular beats (DVBs) which can be used as an indicator of the severity of the AV block. Mane hair Ca, Cu, Mg, and Zn concentrations were measured by the particle-induced X-ray emission method. The Zn/Cu ratio and Ca concentration in mane hair were significantly and positively correlated with the hourly DVBs in horse with a second-degree AV block (p<0.01, r2=0.485; p<0.05, r2=0.351, respectively). Proposed diagnostic cutoff points for hair Ca concentration and Zn/Cu ratio based on receiver operating characteristics curves analysis in detecting second-degree AV block were set at 1536 microg/g and 26.0, respectively. Those results with horse hair suggest that the evaluation of the Ca, Cu, Mg, and Zn status in mane hair by this method is strongly related to the severity of second-degree AV block and might predict the susceptibility of an individual much before the development of the symptom.

Connexin 32 expression reduces malignant phenotype in human A549 adenocarcinoma cells: Implication of Src involvement.

Recent evidence suggests that a member of the gap junction protein family, connexin (Cx) 32, acts as a tumor suppressor gene against lung adenocarcinoma. However, the precise mechanism remains unclear. In this study, we tried to explore the mechanism for the Cx32-dependent tumor-suppressive effect in lung adenocarcinoma. To perform this study, we established a stable clone of the human lung adenocarcinoma cell line, A549 in which the Cx32 gene was expressed. Cx32 expression in A549 cells reduced anchorage-independent growth and development of tumors in a xenograft model. Additionally, Cx32 induced contact inhibition of growth and reduced invasive activity in A549 cells. The tumor-suppressive effects of Cx32 depended on the inhibition of Src activity. These events were confirmed by an Src inhibitor (PP1) and siRNA for Cx32. These results suggest that the Cx32-dependent tumor-suppressive effect in A549 cells is explained by the inhibition of Src activity.

Expression patterns of connexin 26 and connexin 43 mRNA in canine benign and malignant mammary tumours.

The expression patterns of connexin (Cx) genes, encoding gap junctional proteins, are tissue- and cell-specific and, as their expression is mostly suppressed during carcinogenic processes, they are appropriate for monitoring tumour development. In this study, using reverse transcriptase-coupled polymerase chain reaction (RT-PCR), the expression of Cx mRNAs was examined in seven normal canine mammary glands and in 31 mammary gland tumour samples. Cx26 and Cx43 gene expression was studied in all normal tissues using specific Cx26 and Cx43 primers. When the expression patterns of Cx26 and Cx43 genes were analyzed in several types of canine mammary gland tumours, it was noted that it was the loss of Cx26 expression rather than the occurrence of Cx43 expression that was associated with malignancy. These results suggest that Cx26 plays an important role in tumourigenesis of canine mammary gland.

The effect of 7.2% hypertonic saline solution on m-mode echocardiographic indices in normovolemic dogs.

This study investigated whether a small volume of 7.2% hypertonic saline solution (HSS) could affect M-mode echocardiographic indices in dogs. HSS induced significant increase in heart rate, stroke volume and cardiac index, when the fluid infusion was completed (P<0.05). In the HSS group, the left ventricular end-diastolic volume index, as an index of preload, significantly increased (P<0.05), whereas left ventricular end-systolic volume index were not altered. HSS induced slight increases in ejection fraction at end of infusion despite significantly differences were not observed. In conclusion, HSS did not induce a demonstrable effect on M-mode echocardiographic indices of systolic function-enhance cardiac contractility, but it caused preload augmentation that may contribute to an abrupt and transient increase in cardiac output just after HSS infusion.

Relationship between trace elements status in mane hair and atrial fibrillation in horse.

To investigate the relationships between trace elements concentrations in hair and atrial fibrillation (AF) in horses, concentrations of nineteen trace elements were detected in hair using the particle induced X-ray emission (PIXE) method. The horses were assigned to either control (n=22, no abnormalities) or AF groups (n=5) based on electrocardiograph findings. The mean concentrations of Ca and Zn in the hair of the AF group were significantly higher than those in the control group. The Zn/Cu ratio of the hair in the AF group (29.8 +/- 5.5) was significantly higher than that in the control group (23.4 +/- 2.2, P<0.05). The results of the present study suggest that there is a relationship between elevated concentrations of Ca and Zn in hair and AF.

The effect of 7.2% hypertonic saline solution on the duration of sodium gradient between the cerebrospinal fluid and the venous circulation in the dog.

To determine the duration of water movement from cerebrospinal fluid (CSF) into venous blood by the infusion of 7.2% hypertonic saline solution (HSS), the sodium gradient between venous blood and CSF were examined. Venous sodium concentrations remained higher than that in CSF for duration of 60 min following HSS infusion. By 90 min, the CSF sodium concentration reached the equilibrium with venous sodium concentration. Those data suggests that the duration of time during which water moved from CSF into capillaries in brain by the gradient of sodium concentration was less than 90 min.

Peroxisome proliferator-activated receptor delta as a molecular target to regulate lung cancer cell growth.

It has been assumed that prostaglandin (PG)I2 signaling contributes to the negative growth control of lung cancer cells; however, the mechanism remains unresolved. PGI2 functions through a cell surface G protein-coupled receptor (prostaglandin I2-binding receptor, IP) and also exerts an effect by interacting with a nuclear hormone receptor, peroxisome proliferator-activated receptor delta (PPARdelta). We found that PPARdelta was a key molecule of PGI2 signaling to give negative growth control of lung cancer cells (A549), using carbarprostacyclin, a PGI2 agonist for IP and PPARdelta, and L-165041, a PPARdelta agonist. Furthermore, PPARdelta-induced cell growth control was reinforced by the inhibition of cyclooxygenase. These results suggest that PPARdelta activation under the suppression of PG synthesis is important to regulate lung cancer cell growth.

Restoration of connexin 43 by Bowman-Birk protease inhibitor in M5076 bearing mice.

The present study was designed to investigate the effects of Bowman-Birk inhibitor (BBI) on up-regulation of connexin (Cx) expression to estimate BBI's tumor-suppressor effect in mice with M5076 ovarian sarcoma. The relative tumor weight (p<0.05, r(2)=0.301) and proliferating cell nuclear antigen (PCNA, p<0.01, r(2)=0.493) were negatively correlated with the doses of BBI. In contrast, the relative density of Cx43 was positively correlated with the doses of BBI (p<0.05, r(2)=0.351). Therefore, it suggests that the anti-carcinogenic effects of BBI induced negative growth control caused by the expression of Cx43 genes in mice with M5076 ovarian sarcoma.

Optimum dose of LH-RH analogue Fertirelin Acetate for the induction of superovulation in mice.

The optimum dose for establishing superovulation in mice of Fertirelin Acetate (FA), an LH-RH analogue, was examined. Mice were subcutaneously injected with 5 IU of hCG at 17:00 (Day 0), and with various doses of FA (0.001 to 1.0 microg) five times at 4 h intervals on and after 22:00 on Day 0. To induce ovulation, 5 IU of hCG was again injected subcutaneously at 17:00 on Day 2. In the groups administered with doses ranging from 0.01 to 0.5 microg of FA, the number of ovulated eggs was significantly (p<0.05) larger than in the control group (12.9 +/- 5.9). The greatest number of ovulated eggs (22.6 +/- 7.3) was obtained in the group administered with 0.025 microg of FA. The results indicate that the effective dose of LH-RH analogue, FA, is between 0.1 and 0.5 microg for superovulation induction in mice.

Influence of the coat color on the trace elemental status measured by particle-induced X-ray emission in horse hair.

The influence of hair color on the trace elemental status in horse's hair has been studied. A current analytical technique such as particle-induced X-ray emission (PIXE) used in this study has provided reliable, rapid, easy, and relatively inexpensive diagnostic methods. Twenty-eight elements (Al, Br, Ca, Cl, Co, Cu, Cr, Fe, Ga, Hg, K, Mg, Mn, Mo, Na, Nb, Ni, P, Pb, Rb, S, Se, Si, Sr, Ti, V, Y, and Zn) in mane hair were detected by the PIXE method. The gray hair contains significantly greater amounts of Cu, Ti, and Zn, and lower amounts of Br, Ca, Se, and Sr than those in other colored horse hairs (p<0.05). Those results measured in the horse's hair were similar to those found in human and dog hair. When interpreting a result, it should be kept in mind that hair color, especially gray hair, influences the concentrations of some elements in horse hair.

Correlation between 25 element contents in mane hair in riding horses and atrioventricular block.

The influence of atrioventricular block (AV-block) on the trace elemental status in a horse hair was studied. The particle-induced X-ray emission (PIXE) method has provided a reliable, rapid, easy, and relatively inexpensive diagnostic method. Twenty-five elements (Al, Br, Ca, Cl, Co, Cu, Cr, Fe, Hg, K, Mg, Mn, Na, Nb, Ni, P, Pb, Rb, S, Se, Si, Sr, Ti, Y, and Zn) in mane hair and serum were measured by the PIXE method. A horse hair with first- and second-degree AV-block contained significantly greater amounts of Br, Ca, Sr, and Zn than those of horses without electrocardiographic abnormalities, whereas there was no significant differences in the elemental contents of the serum of the both groups. Those results in contents of a horse hair suggest that the evaluation of the degree of ionic imbalance by this method might be used to predict the susceptibility of a horse to heart disease much before symptoms appear.

Twenty-eight element concentrations in mane hair samples of adult riding horses determined by particle-induced X-ray emission.

The concentrations of 28 elements (Al, Br, Ca, Cl, Co, Cu, Cr, Fe, Ga, Hg, K, Mg, Mn, Mo, Na, Nb, Ni, P, Pb, Rb, S, Se, Si, Sr, Ti, V, Y, and Zn) were measured in mane hair by the particle-induced X-ray emission method. Except for Br, Cl, K, S, and P, the trace element concentrations in mane hair of horses are similar to literature values for human hair. The values obtained are not dependent on the horse's age, breed, and sex and could be used as reference values in the assessment of diseases and nutritional status in equines.

Comparison of a small volume of hypertonic saline solution and dextran 40 on hemodynamic alternations in conscious calves.

The hemodynamic effects of rapid intravenous (IV) administration of 10% dextran 40 in saline solution (D40) and 7.2% hypertonic saline solution (HSS) in calves were compared. Calves received isotonic saline solution (ISS), HSS or D40 (3 calves/group) and were monitored of blood pressure, and cardiac output (CO) for 180 min. HSS and D40 infusions induced a significant increase in relative plasma volume reaching 134.9+/-2.8 and 125.0+/-1.9%, respectively at the end of fluid infusion. In the HSS group, CO, cardiac index (CI) and stroke volume (SV) remained constant at low levels after 90 minutes despite the maximal values of CO, CI and SV at the end of infusion, reaching 21.0+/-6.3 l/min (p<0.05), 177.8+/-14.2 ml/min/kg (p<0.001) and 0.20+/-0.03 l/beat (at t=10 min, p<0.001), respectively. In contrast, CI and SV in the D40 group showed significant increases to 14.7+/-2.9 l/min and 153.5+/-17.2 ml/min/kg, respectively, at the end of fluid infusion. And those values remained constant at higher levels than those of the before infusions values throughout the experimental periods. Positive effects for hemodynamic alternations of D40 in calf practice were milder and longer than those of HSS. Therefore, the D40 infusion should be explored as a possible treatment for dehydrated calves, since rapid infusion of D40 may be safe and more beneficial for rehydrating more than HSS treatment.