Oxygen ultra-fine bubbles water administration prevents bone loss of glucocorticoid-induced osteoporosis in mice by suppressing osteoclast differentiation.

Oxygen ultra-fine bubbles (OUB) saline injection prevents bone loss of glucocorti\coid-induced osteoporosis in mice, and OUB inhibit osteoclastogenesis via RANK-TRAF6-c-Fos-NFATc1 signaling and RANK-p38 MAPK signaling in vitro. INTRODUCTION: Ultra-fine bubbles (<200 nm in diameter) have several unique properties, and they are tested in various medical fields. The purpose of this study was to investigate the effects of oxygen ultra-fine bubbles (OUB) on glucocorticoid-induced osteoporosis (GIO) model mice. METHODS: Prednisolone (PSL, 5 mg) was subcutaneously inserted in 6-month-old male C57BL/6J mice, and 200 µl of saline, OUB-diluted saline, or nitrogen ultra-fine bubbles (NUB)-diluted saline was intraperitoneally injected three times per week for 8 weeks the day after operations. Mice were divided into four groups; (1) control, sham-operation + saline; (2) GIO, PSL + saline; (3) GIO + OUB, PSL + OUB saline; (4) GIO + NUB, PSL + NUB saline. The effects of OUB on osteoblasts and osteoclasts were examined by serially diluted OUB medium in vitro. RESULTS: Bone mass was significantly decreased in GIO [bone volume/total volume (%): control vs. GIO 12.6 vs. 7.9; p < 0.01] while significantly preserved in GIO + OUB (GIO vs. GIO + OUB 7.9 vs. 12.9; p < 0.05). In addition, tartrate-resistant acid phosphatase (TRAP)-positive cells in the distal femur [mean osteoclasts number/bone surface (mm-1)] was significantly increased in GIO (control vs. GIO 6.8 vs. 11.6; p < 0.01) while suppressed in GIO + OUB (GIO vs. GIO + OUB 11.6 vs. 7.5; p < 0.01). NUB did not affect these parameters. In vitro experiments revealed that OUB significantly inhibited osteoclastogenesis by inhibiting RANK-TRAF6-c-Fos-NFATc1 signaling, RANK-p38 MAPK signaling, and TRAP/Cathepsin K/DC-STAMP mRNA expression in a concentration-dependent manner. OUB did not affect osteoblastogenesis in vitro. CONCLUSIONS: OUB prevent bone loss in GIO mice by inhibiting osteoclastogenesis.

Feasibility of marrow harvesting from pediatric sibling donors without hematopoietic growth factors and allotransfusion.

We retrospectively studied 108 marrow harvests from 105 pediatric sibling donors. The median age of donors was 8 years (range: 1-15) and the median body weight was 27 kg (range: 10-100). The volumes of aspirated marrow were 5.0-23.8 mL/kg donor body weight, and harvested bone marrow volume exceeded 15 mL/kg in 42% of the donors. A total of 100 autologous blood donations were performed, and eight donors had red cells salvaged from their harvests reinfused. The median Hb levels before and after harvests were 12.3 g/dL (range: 10.0-14.7) and 11.0 g/dL (range: 8.9-13.8), respectively. None of the donors received allogeneic blood transfusions or hematopoietic growth factors such as EPO and G-CSF before or after collection. Transplanted dose was 1.4-10.8 × 10(8) cells/kg recipient body weight without differences due to donor age. Higher concentrations of nucleated and CD34(+) cells were obtained from younger donors. All donors tolerated the procedures well, with no serious complications. Thus, children may safely donate marrow for allogeneic transplantation, and the yields of nucleated cells for engraftment are substantial.

Association between patient education and health-related quality of life in patients with Parkinson's disease.

BACKGROUND: Providing patients with disease- and treatment-related information is an important role of medical staff and is now reimbursed in Japan by the national health insurance system under the rubric 'patient education'. Evaluation of the effectiveness of patient education programs is necessary to ensure that limited health care resources are used efficiently. OBJECTIVE: The objective is to determine whether educating patients with Parkinson's disease (PD) is related to better health-related quality of life (HRQOL). DESIGN: A cross-sectional study was conducted. SETTING: Members of the Japan Association of Patients with Parkinson's disease were randomly selected. PARTICIPANTS: A total of 1200 patients with PD were asked to fill in written questionnaires and replies from 762 (63.5%) were analyzed. MEASUREMENTS: The questionnaire inquired about clinical characteristics, comorbidity, symptoms of PD, complications of therapy, HRQOL, and patient education. SF-36 was used to assess HRQOL. The section on patient education comprised one question each on patient-perceived satisfaction with information provided on (1) disease condition and pathophysiology, (2) effectiveness of drug therapy, (3) adverse drug reactions, (4) publicly available financial and social resources, and (5) rehabilitation and daily activities. Patient education score was defined as the sum of the individual scores for these five questions. The relationships between scores on the SF-36 subscales and the patient education score were examined. RESULTS: More satisfaction with patient education was associated with higher scores in all SF-36 subscales except physical functioning and bodily pain. The difference in score between the most satisfied and the least satisfied patients ranged from 8.4 points on the subscales of general health and 16.7 points on the subscale of role limitation due to emotional problems. CONCLUSION: The conclusion that patient education is associated with better HRQOL in patients with PD is drawn.

Isolation, expression and characterization of carp retinol-binding protein.

Vitamin A alcohol and its precursors carotenoids are introduced in the organism with the diet, transported to the liver and from there as retinol to target tissues by a specific carrier, the retinol-binding protein (RBP). RBP, isolated and characterized in many vertebrates, shows very high homology among the species investigated; however, very little is known in fish. In the present work RBP cDNA isolated from a carp liver library was transcribed and translated in vitro and the corresponding protein characterized. Carp RBP amino acid sequence and tertiary structure are highly conserved, but the protein shows two peculiar and unique characteristics: the signal sequence is not processed by the ER signal peptidase and two N-glycosylations are present at the N-terminus portion of the protein. It was also demonstrated that RBP glycosylation is not a feature common to all teleosts. Transfection experiments show that the green fluorescent protein (GFP) can be directed into the secretory pathway by the carp RBP N-terminal region, both in fish and in mammal cells, demonstrating that the sequence, although not processed, is recognized as a secretory signal in different species. Results obtained from different investigators indicated that in fish plasma RBP circulates without interacting with transthyretin (TTR) or other proteins, suggesting that the complex with TTR, whose postulated function is to hamper easy kidney filtration of circulating RBP, has evolved later in the evolutionary scale. This hypothesis is reinforced by the finding that carp RBP, as well as trout and other lower vertebrates in which circulating complex has never been demonstrated, lacks a short C-terminal sequence that seems to be involved in RBP-TTR interaction. In carp, carbohydrates could be involved in the control of protein filtration through the kidney glomeruli. Moreover, experiments of carp RBP expression in Cos-1 cells and in the yeast Saccharomyces cerevisiae show that glycosylation is necessary for protein secretion; in particular, additional in vitro experiments have shown it is involved in protein translocation through ER membranes.

Unique biochemical nature of carp retinol-binding protein. N-linked glycosylation and uncleavable NH2-terminal signal peptide.

Retinol transport and metabolism have been well characterized in mammals; however, very little is known in fish. To study the mechanism by which fish retinol-binding protein (RBP) is able to remain in plasma besides its small molecular size, we isolated RBP cDNA from a carp liver cDNA library. Comparison of the deduced amino acid sequence with that of known vertebrate RBPs showed that carp RBP has high homology to the other cloned vertebrate RBPs, but it lacks the COOH-terminal tetrapeptide, RNL(S)L, which is most likely involved in the interaction with transthyretin in mammalian RBPs. In addition, the primary structure of carp RBP contains two consensus N-linked glycosylation sites that represent a unique feature. We have obtained experimental evidence, by in vitro and in vivo expression experiments, that both sites are indeed glycosylated. We have also characterized the protein as a complex type N-linked glycoprotein by lectin binding assay, neuraminidase and endoglycosidase H and F digestion. Inhibition of glycosylation by tunicamycin treatment of transfected cells caused a great reduction of RBP secretion. Since kidney filtration of anionic proteins is less than half that of neutral protein of the same size, this finding strongly suggests that the amount of carp RBP filtration through kidney glomeruli may be reduced by a glycosylation-dependent increase in the molecular size and negative charge of the protein. A second unique feature of carp RBP as secretory protein is the presence of a nonconserved NH(2)-terminal hydrophobic domain, which functions as an insertion signal but is not cleaved cotranslationally and remains in the secreted RBP.

The effect of pentylenetetrazole-kindling on the extracellular glutamate and taurine levels in the frontal cortex of rats.

We studied the extracellular concentration of glutamate and taurine in the frontal cortex of freely-moving pentylenetetrazole (PTZ) kindled rats using an in vivo microdialysis. A significant and sustained increase in the glutamate level was observed in the kindled rats, in contrast, a slight and delayed increase was observed in the non-kindled rats when the same grade seizure was induced by PTZ. The convulsive dose of PTZ administration caused a decrease in taurine levels in the controls, however, no significant changes were found in the kindled rats.

Dorsal column-thalamic pathway is involved in thalamic hyperexcitability following peripheral nerve injury: a lesion study in rats with experimental mononeuropathy.

A total of 68 neurons were recorded from the ventro-postero-lateral nucleus of thalamus (VPL) in rats with a unilateral chronic constriction injury (CCI) of the sciatic nerve (n=20), sham operation (n=24) and naive rats (n=24), and effects of the lesion of dorsal column (DC) pathway [DC lesion or DC+gracile nucleus lesions] on VPL nucleus neuronal activities were studied. In the VPL nucleus contralateral to the CCI (receiving input from the injured nerve), response latencies of low threshold mechanoreceptive (LTM) and wide dynamic range (WDR) neurons to electrical stimulation of the sciatic nerve were significantly longer than that in the contralateral VPL nucleus receiving input from the sham-operated side (P<0.05). In contrast, response latencies of LTM and WDR neurons to DC stimulation were not different between the sham operated and CCI sides (0.05). Background activity of WDR neurons was significantly higher in the VPL nucleus contralateral to the CCI side when compared to neurons in the VPL nucleus contralateral to the sham operated side and in naive animals. Responses of LTM and WDR neurons to innocuous mechanical stimulation of the receptive fields were significantly decreased after DC and DC+gracile nucleus lesions in all animals. However, the responses of WDR neurons to noxious stimuli were selectively reduced only in rats with CCI by DC and DC+gracile nucleus lesions (P<0.05). The decrease in noxious stimulus-evoked responses of WDR neurons in the VPL nucleus contralateral to the CCI side after DC and DC+gracile nucleus lesions was greater than that in the VPL nucleus contralateral to the sham operated side and naive animals. These results indicated that DC and DC+gracile nucleus lesions produced selective and stronger effect on noxious responses of VPL nucleus WDR neurons receiving input from the site of nerve injury. The findings suggest that the gracile nucleus-thalamic pathway conveys, or modulates, nociceptive information to the VPL nucleus following peripheral nerve injury, resulting in an increase in VPL nucleus response to noxious stimuli that contributes to the development of mechanical hyperalgesia.

[Evaluation of intermittent hepatic arterial infusion chemotherapy for multiple liver metastasis of colorectal cancer].

Five patients with synchronous multiple hepatic metastasis of colorectal cancer were treated with hepatic arterial infusion chemotherapy. All cases received intermittent 5-FU infusion (5-FU 250-1,000 mg/2-3 hrs/1-2 weeks) on an outpatient basis. In the evaluation of 5 cases, 3 PR and 1 NC were observed. One case administered arterial infusion for adjuvant chemotherapy has no recurrence in liver. In two patients, extra-hepatic metastases were found. In conclusion, this therapy was effective and useful for hepatic metastasis. Moreover, other forms of treatment for extra-hepatic metastasis must be used.

[The effect of Shakuyaku-kanzo-to on prostaglandin production in human uterine myometrium].

Shakuyaku-kanzo-to (SK), a Chinese herb medicine consisting of Shakuyaku (Paeoniae Radix) and Kanzo (Glycyrrhizae Radix) has been used for the treatment of dysmenorrhea. It is reported that prostaglandin (PG) production increased during menstruation in uterine myometrium. To know the effect of SK on PG production in the uterine myometrium, the following in vitro study was undertaken. Human uterine myometrium was obtained from patients who underwent hysterectomy. 1. Myometrial cells were cultured and, SK, Shakuyaku (S), Kanzo (K), or glycyrrhetinic acid (GA), which is the major component of K, were added to the culture medium. Concentrations of PGE2, PGF2 alpha, and 6-ketoPGF1 alpha in the medium measured by RIA were significantly decreased by the addition of SK, K or GA but no effect was observed when S. 2. [3H]-arachidonic acid (AA) was added to culture medium and incorporated into the Sn-2 of phospholipids. [3H]-AA release and PG production in the medium were determined. PG were extracted and the radioactivity of PG was measured. The production of PGE2, PGE2 alpha and 6-ketoPGF1 alpha from labeled cells was significantly reduced by the addition of SK, K and GA. 3. Phosphatidylcholine containing [14C]-AA in Sn-2 (150,000 dpm) was incubated with cytosol of uterine myometrium and the amounts of [14C]-AA released were calculated as Phospholipase A2 activity. The amount of [14C]-AA release was inhibited dose-dependently by SK, K and GA. It is the first time that SK has been shown to suppress PG production in the myometrium by inhibiting cPLA2 activity.

Only the first and the last hydrophobic segments in the COOH-terminal third of Na,K-ATPase alpha subunit initiate and halt, respectively, membrane translocation of the newly synthesized polypeptide. Implications for the membrane topology.

We studied the topogenic properties of five hydrophobic segments (H5-H9) in the COOH-terminal third of Na,K-ATPase alpha subunit using in vitro insertion of fusion proteins into endoplasmic reticulum membranes. These fusion proteins consisted of several different lengths of truncated alpha subunit starting at Met729 and a reporter protein, chloramphenicol acetyltransferase, that was linked in frame after each hydrophobic segment. We found that membrane insertion of the newly synthesized COOH-terminal third was initiated by H5 and terminated by H9, indicating that here only H5 and H9 have topogenic function. The other three, H6-H8, did not have topogenic function in the native context and were translocated into the endoplasmic reticulum lumen. These results were in striking contrast to the previous models in which four or six hydrophobic segments were proposed to cross the membrane. Furthermore, the findings suggest a novel mechanism for achieving the final membrane topology of the COOH-terminal third of the alpha subunit.

The pathogenic role of the NMDA receptor in hyperthermia-induced seizures in developing rats.

Hyperthermia-induced seizures (HS) in rats have been used as a model of febrile seizures. Activation of the N-methyl-D-aspartate (NMDA) receptor by increased extracellular glutamate (Glu) in the cortex during hyperthermia may be involved in the induction of HS and HS kindling. To confirm this hypothesis, the effects of a potent blocker of the NMDA receptor, MK-801, on the threshold and pattern of HS were evaluated. The threshold temperatures for rats given 0.1 (low dose) and 0.5 (high dose) mg/kg MK-801 (i.p.) for the first time were 41.6 degrees C (39.7-42) (median, range) and 42.0 (41.2-42.0), respectively, which were significantly higher than the 40.5 (39.4-41.2) for rats without MK-801 administration (P < 0.01). The recurrent occurrence of HS suppressed the increase in the threshold temperature with age, and changed the seizure from partial to generalized seizures (HS kindling), whereas these effects of recurrent HS on the threshold and pattern of HS were inhibited by the high dose (0.5 mg/kg) of MK-801. MK-801 blocks HS and HS kindling. The activation of the NMDA receptor during hyperthermia plays an important role in the induction of HS and HS kindling.

An experimental study of conductive heating using a concentric double-electrode applicator.

With hyperthermia for treatment of superficial tumors in mind, a prototype applicator with two electrodes arranged concentrically on a disk was designed for efficient local heating, and a basic heating test was carried out. Frequencies as low as 200 kHz were used in order to simplify the configuration of the power device. The applicator consists of two electrodes, a circular inner electrode and another looped outer electrode, arranged concentrically. Water was passed through the applicator as a cooling mechanism; it was placed in direct contact with the target tissue to be heated and then charged with electricity. In the heating test using a phantom, oval hot spots were noted below the inner electrode. Using cooling water at 3 degrees C and 8.2 W, an isothermal line of 45 degrees C was located at a 5-mm radius circle around the central axis with 9 mm depth. A similar temperature distribution map was obtained in heating tests on the thigh muscle of a mongrel adult dog. The temperature distribution maps obtained from these tests corresponded closely with the results of theoretical analysis carried out according to the finite-element method. Since a comparatively low frequency was employed for this applicator the power device was simplified, which made adequate heating possible with low electric power. The temperature distribution map indicated that efficient local heating of superficial tumors could be achieved.

[Effects of mosapramine (Y-516), a new dopamine D2 antagonist, on reverse tolerance after repeated administration of methamphetamine by means of the ambulation-increasing effect in mice].

Effects of mosapramine (Y-516), a new dopamine D2 antagonist, on reverse tolerance (sensitization) after repeated administration of methamphetamine (MAP; 2 mg/kg, s.c.) were investigated by means of ambulatory activity in mice; and they were compared with those of clocapramine (CCP), bromperidol (BPD) and chlorpromazine (CPZ). Y-516 (0.3, 1, 3 and 10 mg/kg, p.o.), CCP (3, 10 and 30 mg/kg, p.o.), BPD (0.1, 0.3 and 1 mg/kg, p.o.), CPZ (1, 3 and 10 mg/kg, p.o.) or 0.5% methylcellulose (MC; solvent, p.o.) were given to mice 30 min before MAP administration. The ambulatory activity was measured by tilting-type activity changes for 3 hr after MAP. These treatments were repeated 5 times at 3-4 day intervals. Then MAP alone was challenge-administered to all of these mice 3-4 days after the final administration. Marked reverse tolerance was produced after repeated administration of MC plus MAP. On the other hand, the ambulation-increasing effect of MAP was suppressed dose-dependently in groups pretreated with Y-516 or comparison-drugs, although the development of reverse tolerance was not completely inhibited after the repeated administration. In the challenge-administration of MAP, the ambulation-increasing effect was dose-dependently suppressed in the Y-516 group or the comparison-drug plus MAP group as compared with that in the MC plus MAP group.

[Pharmacokinetical and clinical study of cefpirome in children].

This study describes the pharmacokinetic characteristics and clinical usefulness of cefpirome (CPR) in children. Mean half-lives of 20 mg/kg and 40 mg/kg of CPR injected intravenously in one shot were 1.18 and 1.34 hours, respectively, and their mean recovery rates into urine were 69.8 and 72.2%, respectively. Minimum inhibitory concentrations of CPR against Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli and Haemophilus influenzae were the same as or lower than those of ceftazidime. CPR was clinically effective in 14/15 of patients with bacterial infections; 8/9 of pneumonia, 2/2 of bronchitis, 1/1 of pharyngitis, 1/1 of tonsillitis, 1/1 of osteomyelitis, 1/1 of urinary tract infection. No clinically overt side effects of CPR were found, while an increase of eosinophils in blood was observed in 2 cases, and an increase of platelet in blood in 1 case and an elevation of serum GPT activity in 1 case were also observed. These findings indicate that CPR is useful for the treatment of bacterial infections in children.

[Evaluation on equivalence of pharmacological properties between natural crude drugs and their cultured cells based on their components. Accelerative effect of lithospermi radix and inhibitory effect of coptidis rhizoma on proliferation of granulation tissue].

The present study was carried out to evaluate an equivalence of pharmacological properties between natural crude drugs and their cultured cells. The effects of ether extract of Lithospermi Radix and cultured cells of Lithospermum erythrorhizon Sieb. et Zucc. and aqueous extract of Coptidis Rizoma and cultured cells of Coptis japonica Makino var. dissecta Nakai on proliferation of granulation tissue in rats were compared. The ether extracts of Lithospermi Radix and the cultured cells enhanced proliferation of granulation tissue by the cotton pellet method. The potency of both extract was about the same, if results were compared with the corresponding doses which contained the same quantity of shikonin derivatives. On the other hand, the aqueous extracts of Coptidis Rhizoma and the cultured cells inhibited it. The potency of both extract was about the same, if results were compared with the corresponding doses which contained the same quantity of berberine-type alkaloids. From these results, to evaluate an equivalence of pharmacological properties between natural crude drugs and their cultured cells, it is concluded that their qualities and quantities are not so different each other and the almost same pharmacological effect expected on the basis of their uses is required.

Hyperthermia-induced seizures with a servo system: neurophysiological roles of age, temperature elevation rate and regional GABA content in the rat.

A servo system including a microwave generator was applied to raise a rat's body temperature at a pre-set rate. Using this system the effects of age and the temperature elevation rate upon febrile seizures in rats were studied. The relationship between the brain GABA content and hyperthermia was also studied. From the results of the present study, the seizure occurrence rate was found to be highest at the age of 20 days, brain damage was speculated to be severe after hyperthermia-induced seizures with a slow temperature rise, and the regional GABA concentration in subcortical structures was found to increase during hyperthermia. These data indicate that a servo system with a microwave generator is useful for experimental febrile convulsions, and that GABA neurotransmission in subcortical structures might contribute to feed-back regulation against seizures during hyperthermia.

Flow Cytometric Analysis of Cellular Berberine Contents in High- and Low-Producing Cell Lines of Coptis japonica Obtained by Repeated Selection.

Cell lines that were highly productive for berberine were selected by repeated cloning of small cell aggregates. The berberine content of the highest-producing cell line increased after 4 clonings to 10% dw in comparison to the 3% dw found for the parent line, and the berberine yield was about 1,500 mg/1/14 days. There was no increase in berberine yield after the fifth cloning. Low-producing cell lines also appeared, even as the progeny of a highly productive cell line. We investigated the function of clonal selection in the enhancement of cellular berberine production. Flow cytometric analysis showed that high- and low-producing cell lines gave the fluorescence derived from the berberine contents of individual cells over essentially the same range of fluorescent intensity; but, the mode of fluorescence distribution shifted to a higher intensity with the increase in the berberine content of a cell line. This enhancement of berberine production because of cell selection must, therefore, be caused by a number of cells in a population that have high alkaloid contents, not by a uniform increase in the berberine content of all the cells.

[A randomized controlled trial of surgical adjuvant therapy with mitomycin C, 5-fluorouracil and OK-432 in patients with gastric cancer].

The effect of postoperative immunochemotherapy with mitomycin C (MMC), 5-fluorouracil (5-FU) and OK-432 was evaluated as an adjuvant therapy after curative resection for gastric cancer. Immediately after surgery, patients were randomly allocated to the following three treatments: (A) chemotherapy with MMC and 5-FU (32 cases); (B) chemoimmunotherapy with MMC, 5-FU and OK-432 (33 cases); and (C) surgery alone as control (34 cases). There were no significant differences in the background factors influencing survival time among the groups, and there was no dose-distribution of chemotherapeutic agents between groups A and B. While the differences were not statistically significant, the survival rate and disease-free interval of group B were better than those of groups A or C. Side effects such as gastroenteric disorder, leukopenia (less than 3,000/mm3), thrombocytopenia (less than 7 X 10(4)/mm3) and increase of serum transaminase level (GPT greater than or equal to 100 units) were less frequently observed in group B than in group A. The results of the present study seemed to indicate that chemoimmunotherapy with OK-432 may be effective for surgical adjuvant therapy.

Intracerebroventricular injection of 125I-salmon calcitonin in rats: fate, anorexia and hypocalcemia.

Intracerebroventricular (i.c.v.) injection of 19 pmol/rat or more of salmon calcitonin (sCT) or iodinated sCT suppressed spontaneous intake of food and water in a dose-dependent manner. Tail-whipping was a peculiar behavior which concomitantly developed, but no analgesia ensued from the doses tested (up to 62 pmol/rat). It was examined how the rise and fall pattern of these behavioral effects would correlate with the dispositional pattern of 125I-sCT. When the radioactive peptide was injected in anorectic doses via the i.c.v. route, the radioactivity was found to distribute throughout the brain, but not uniformly. In rats which showed a marked anorexia and tail-whipping behavior, distribution occurred in such a manner that it could be interpreted to reflect the regional and subcellular distribution pattern of sCT-specific binding sites. Even 3 hr after injection, the hypothalamus, the smallest region, retained the highest radioactivity corresponding to about 1% of the dose and at least one half of which was identified as the intact iodo-sCT. To be noted is the finding that sCT injected centrally will quickly enter the systemic circulation and peripherally induced long-lasting hypocalcemia, since the anorectic dose of sCT is considerably higher than the dose needed for the peripheral effect. It is concluded that most of the sCT after i.c.v. injection leaks into the systemic circulation, but the rest is retained rather selectively around the receptor in hypothalamic nuclei for a long time, leading to day-long suppression of feeding and drinking behavior.