Comparison of Residual Dimethyl Sulfoxide (DMSO) and Ethylene Glycol (EG) Concentration in Bovine Ovarian Tissue During Warming Steps Between Slow Freezing and Vitrification.

BACKGROUND: DMSO and EG have been used as cryoprotectants for human ovarian tissue cryopreservation, but residual cryoprotectants concentration and safety have rarely been reported. OBJECTIVE: We aimed to compare residual cryoprotectants (DMSO, EG) concentration in bovine ovarian tissue during warming steps between one kind of common slow freezing method and two kinds of vitrification methods, which are usually used for cryopreservation of human ovarian tissue in Japan. MATERIALS AND METHODS: In this study, we used five bovine ovaries with an average age of 24.2 months divided into three kinds of cryopreservation methods. All ovarian cortices cut to 1 mm thickness were cryopreserved in slow freezing and two kinds of vitrification methods. Residual cryoprotectants before, during and after warming of cryopreserved ovarian cortices were measured using GC-MS and compared. RESULTS: Concentrations of residual cryoprotectants in the ovarian tissue just before transplantation into the body after warming were high after both vitrification methods but almost zero with the slow freezing method. CONCLUSION: We are concerned about the residual cryoprotectants in ovarian tissue, and continue to study the safety of cryopreservation methods to the woman after reimplantation and her baby.

A transportation network for human ovarian tissue is indispensable to success for fertility preservation.

PURPOSE: The purpose of this study was to examine the efficacy of an ovarian tissue transportation network for fertility preservation (FP) for cancer patients in Japan. METHODS: PubMed was searched for papers on transportation of human ovarian tissue for FP. We analyzed population, area, number of cancer patients for ovarian tissue cryopreservation (OTC), quality control/assessment and safety, cost of a cryopreservation center for the building for 30 years, and medical fees of cancer patients (operation, cryopreservation, and storage of ovarian tissue). RESULTS: More than twenty babies have been born in Denmark and Germany through a transportation system. Up to 400 new patients a year need OTC. The fees for removal, cryopreservation, and storage for 5 years, and transplantation of ovarian tissue are around €5,000, €4,000, and €5,000, respectively. It costs more than €5 million to establish and maintain one cryopreservation center for 30 years. If we have a few cryopreservation centers in Japan, we can cryopreserve 400 patients' ovarian tissue per year by safer slow freezing and maintain quality control/assessment. We need to lighten the patients' burden for easy to use FP by a government subsidy and medical insurance coverage. CONCLUSIONS: This model has been termed the Danish model ("the woman stays - the tissue moves"). This is truly patient-centered medicine. We can have maximum effects with the minimum burden. A transportation network like those of Denmark and Germany is the best strategy for FP in Japan. It may be the best system for cancer patients, medical staff, and the Ministry of Health, Labor, and Welfare.

Fatty acid composition of lipids in day 7-13 blastocysts, serum and uterine fluid of rabbits.

Purpose: The fatty acid composition of rabbit blastocysts, blood serum and uterine fluids were analyzed to study embryonic lipid metabolism. Methods: Embryos were collected from Japanese white rabbits and fatty acids were analyzed by gas chromatograph. Results: Total amount of fatty acids in blastocysts was higher than that in serum and uterine fluid. The amount of fatty acids in blastocysts markedly decreased during days 7-13 of pregnancy, and in serum had hovered, but in uterine fluid on day 13 was nine times higher than that on day 7 of pregnancy. Palmitic acid predominates in blastocysts, serum and uterine fluid during this period. Conclusion: Palmitic acid is the most abundant fatty acid in the blastocysts, serum and uterine fluids of rabbit during days 7-13 of pregnancy.

Triamcinolone acetonide suppresses early proangiogenic response in retinal pigment epithelial cells after photodynamic therapy in vitro.

OBJECTIVE: To investigate the expression of proangiogenic and antiangiogenic factors, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in retinal pigment epithelial (RPE) cells after photodynamic therapy (PDT), especially focusing on their change in the presence of triamcinolone acetonide. METHODS: Firstly, the cellular uptake of verteporfin was quantified after confluent ARPE-19 (human retinal pigment epithelial) cells were exposed to 5 microg/ml verteporfin combined with or without 1 microg/ml triamcinolone acetonide for 1 h. Secondly, ARPE-19 cells exposed to various doses of verteporfin were irradiated with 120 mJ/cm(2) light. After incubation with or without 1 microg/ml triamcinolone acetonide for 2 days, cell viability and expressions of VEGF and PEDF were assessed. RESULTS: Cellular uptake of verteporfin was not significantly changed by the presence of 1 microg/ml triamcinolone acetonide. In addition, 0.01-0.1 microg/ml of verteporfin showed a dose-dependent toxicity on the ARPE-19 cells 2 days after the light exposure. The presence of verteporfin at a concentration of 0.01 microg/ml did not affect the cell viability but significantly increased VEGF (p<0.001) and reduced PEDF (p = 0.03) expression. Administration of triamcinolone acetonide significantly suppressed both this increase in VEGF (p<0.001) and decrease in PEDF (p = 0.001). CONCLUSIONS: VEGF was increased and PEDF reduced in cultured RPE cells shortly after PDT even at a sublethal dose. Triamcinolone acetonide suppressed this proangiogenic response.

Retinal degeneration is delayed by tissue factor pathway inhibitor-2 in RCS rats and a sodium-iodate-induced model in rabbits.

PURPOSE: To investigate the in vivo effects of tissue factor pathway inhibitor 2 (TFPI-2), which stimulates proliferation of retinal pigment epithelial cells, but not the proliferation of fibroblast and vascular endothelial cells in vitro, on retinal degeneration using a sodium-iodate (SI)-induced model in rabbits and Royal Collage of Surgeons (RCS) rats. METHODS: 79 microg of recombinant TFPI-2 (rTFPI-2) or vehicle alone was injected intravitreously to 18 eyes of 12 pigmented rabbits a day after 20 mg/kg of SI was intravenously administered. Retinal function was assessed 4, 7, 14, and 21 days after the injection by analysing amplitudes of the c-wave of a bright flash electroretinogram. Additionally, 10 microg of rTFPI-2 or vehicle alone was injected intravitreously to 11 eyes of RCS rats at both 3 and 4 weeks old, then the retina was examined histologically at 5 weeks old. RESULTS: The rTFPI-2-treated eyes in rabbits showed a significantly less decrease in the relative amplitude of the c-wave than control eyes on days 4 and 7. The thickness of the outer nuclear layer was significantly thicker and the vacuole in the photoreceptor layer was less frequently observed in the rTFPI-2-treated RCS rats than the controls. CONCLUSIONS: Intravitreal injection of TFPI-2 rescues SI-induced retinal degeneration in rabbits and naturally occurring retinal degeneration in RCS rats at least partly. These results may suggest that this compound can be utilized in the treatment of retinal degeneration.

Transpupillary thermotherapy for treatment of exudative age-related macular degeneration in Japanese patients.

PURPOSE: To report the therapeutic outcome of transpupillary thermotherapy (TTT) for subfoveal choroidal neovascularizarion (CNV) in brown retina using a diode-laser with the setting of lower energy level compared to the previous studies on light-pigmented Caucasian patients. METHODS: A total of 19 subfoveal CNVs in 18 patients were treated with TTT. The power of diode-laser was set 160 mW for 1.2 mm beam, 270 mW for 2.0 mm beam, and 400 mW for 3.0 mm beam, and the laser was delivered for 1 min through a slit-lamp mounted-delivery system. Patients were followed up for a mean of 8.8 months (4-12 months). Visual acuity and the fundus change as judged by funduscopic examination and simultaneous fluorescein and indocyanine green angiography were evaluated. Visual acuity was measured by a Japanese standard Landolt visual acuity chart and converted to logarithm of the minimal angle resolution (log MAR) visual acuity for statistical analysis. Improvement or decline in vision was defined as change of more than 0.2 in log MAR visual acuity. RESULTS: In eyes with minimally classic or occult only CNV, visual acuity improved in two eyes (18%) stabilized in seven eyes (64%) and worsened in two eyes (18%). In eyes with predominantly classic CNV, visual acuity improved in two eyes (25%), stabilized in four eyes (50%) and worsened in two eyes (25%). In all, 15 (84%) eyes of all studied subjects had improvement in exudation. Two (11%) and one (5%) eye(s) were noted to have a significant post-treatment haemorrhage and retinal pigment epithelial tear, respectively. CONCLUSION: In patients with brown retinal colour, the treatment outcome of TTT was comparable to that of light-pigmented Caucasian patients with approximately half the laser power energy. Further randomized control studies are warranted.

The effects of prolonged low-flow sevoflurane anesthesia on renal and hepatic function.

UNLABELLED: We assessed the effects of prolonged low-flow sevoflurane anesthesia on renal and hepatic functions by comparing high-flow sevoflurane with low-flow isoflurane anesthesia. Thirty patients scheduled for surgery of > or =10 h in duration randomly received either low-flow (1 L/min) sevoflurane anesthesia (n = 10), high-flow (6-10 L/min) sevoflurane anesthesia (n = 10), or low-flow (1 L/min) isoflurane anesthesia (n = 10). We measured the circuit concentrations of Compound A and serum fluoride. Renal function was assessed by blood urea nitrogen, serum creatinine, creatinine clearance, and urinary excretion of glucose, albumin, protein, and N:-acetyl-beta-D-glucosaminidase. The hepatic function was assessed by serum aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, and total bilirubin. Compound A exposure was 277 +/- 120 (135-478) ppm-h (mean +/- SD [range]) in the low-flow sevoflurane anesthesia. The maximum concentration of serum fluoride was 53.6 +/- 5.3 (43.4-59.3) micromol/L for the low-flow sevoflurane anesthesia, 47.1 +/- 21.2 (21.4-82.3) micromol/L for the high-flow sevoflurane anesthesia, and 7.4 +/- 3.2 (3.2-14.0) micromol/L for the low-flow isoflurane anesthesia. Blood urea nitrogen and serum creatinine were within the normal range, and creatinine clearance did not decrease throughout the study period in any group. Urinary excretion of glucose, albumin, protein, and N:-acetyl-beta-D-glucosaminidase increased after anesthesia in all groups, but no significant differences were seen among the three groups at any time point after anesthesia. Lactate dehydrogenase and alkaline phosphatase on postanesthesia Day 1 were higher in the high-flow sevoflurane group than in the low-flow sevoflurane group. However, there were no significant differences in any other hepatic function tests among the groups. We conclude that prolonged low-flow sevoflurane anesthesia has the same effect on renal and hepatic functions as high-flow sevoflurane and low-flow isoflurane anesthesia. IMPLICATIONS: During low-flow sevoflurane anesthesia, intake of Compound A reached 277 +/- 120 ppm-h, but the effect on the kidney and the liver was the same in high-flow sevoflurane and low-flow isoflurane anesthesia.

Synthetic conversion of ACAT inhibitor to acetylcholinesterase inhibitor.

Natural product acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor pyripyropene A was synthetically converted to acetylcholinesterase (AChE) inhibitor via heterolitic cleavage of the 2-pyrone ring, followed by gamma-acylation/cyclization with several aroyl chlorides. The 4-pyridyl analogue selectively showed AChE inhibitory activity (IC50 7.9 microM) and no ACAT inhibitory activity IC50 = >1000 microM.

[The compound A concentration in a low-flow anesthesia circuit using the new CO2 absorbent SPHERASORB].

CO2 absorbents convert sevoflurane to fluoromethyl-2,2-difluoro-1-(trifluoromethyl) vinyl ether (compound A), whose toxicity in rats raises concern regarding the safety of sevoflurane in a low-flow system. SPHERASORB is a new CO2 absorbent which does not contain KOH. However, the reaction between SPHERASORB and sevoflurane has not been examined. We compared compound A concentration in a model circuit using SPHERASORB and the commonly used Sodasorb II. The anesthesia circuit was circulated with fresh gas flow at a rate of 1 l.min-1 containing 2% sevoflurane. Compound A concentration was measured hourly and the temperature of CO2 absorbent was monitored. The maximum concentration of compound A in the circuit was 12.2 +/- 1.4 ppm for SPHERASORB and 18.6 +/- 0.4 ppm for Sodasorb II (P < 0.05). The maximum temperature of SPHERASORB was lower than that of Sodasorb II (P < 0.05). SPHERASORB can reduce compound A formation, compared to Sodasorb II.

Reversal of multidrug resistance by 7-O-benzoylpyripyropene A in multidrug-resistant tumor cells.

7-O-Benzoylpyripyropene A (7-O-BzP), a semi-synthetic analog of pyripyropene, was investigated for its reversing effect on multidrug-resistant (MDR) tumor cells. 7-O-BzP (6.25 microg/ml) completely reversed resistance against vincristine and adriamycin in vincristine-resistant KB cells (VJ-300) and adriamycin-resistant P388 cells (P388/ADR), respectively. 7-O-BzP alone had no effect on the growth of drug sensitive and drug-resistant cells. 7-O-BzP (6.25 microg/ml) significantly enhanced accumulation of [3H]vincristine in VJ-300 cells and completely inhibited the binding of [3H]azidopine to the P-glycoprotein in VJ-300 cells and P388/ADR cells. The result suggests that 7-O-BzP effectively reverses P-glycoprotein-related MDR by interacting directly with P-glycoprotein in drug resistant VJ-300 and P388/ADR cells.

Rhabdomyolysis in association with Duchenne's muscular dystrophy.

PURPOSE: To present a case of rhabdomyolysis which developed in a child with a known history of Duchenne's muscular dystrophy, following an anesthetic which included sevoflurane. CLINICAL FEATURES: An 11 yr old boy with a known history of Duchenne's muscular dystrophy underwent anesthesia for strabismus repair. The anesthetic consisted of sevoflurane and nitrous oxide without the use of a muscle relaxant. His postoperative course was complicated by a complaint of heel pain and the development of myoglobinuria. He was treated with dantrolene sodium and discharged home after two days, without further complication. CONCLUSION: Sevoflurane anesthesia has not been shown previously to be associated with the development of acute rhabdomyolysis in a child with a history of Duchenne's muscular dystrophy. As with halothane and isoflurane, the continued use of sevoflurane in the presence of Duchenne's muscular dystrophy should be questioned.

Chemical modification and structure-activity relationships of pyripyropenes. 3. Synthetic conversion of pyridine-pyrone moiety.

Structure-activity relationships of the pyridine-pyrone moiety in pyripyropene A (1), a potent acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor of fungal origin, were studied. Several kinds of aromatic or hetero ring substituents for the pyridine moiety were synthesized using unique degradation reaction, following by gamma-acylation. All the six synthesized analogs decreased the inhibitory activity with 20 to 200 times larger IC50 values than that of 1. Furthermore, the pyridine-pyrone substituent also dramatically decrease the inhibitory activity. Thus, the pyridine-pyrone moiety is important for eliciting potent ACAT inhibition.

Chemical modification and structure-activity relationships of pyripyropenes. 3. Synthetic conversion of pyridine-pyrone moiety

Structure-activity relationships of the pyridine-pyrone moiety in pyripyropene A (1), a potent acyl-CoA : cholesterol acyltransferase (ACAT) inhibitor of fungal origin, were studied. Several kinds of aromatic or hetero ring substituents for the pyridine moiety were synthesized using unique degradation reaction, following by gamma-acylation. All the six synthesized analogs decreased the inhibitory activity with 20 to 200 times larger IC50 values than that of 1. Furthermore, the pyridine-pyrone substituent also dramatically decrease the inhibitory activity. Thus, the pyridine-pyrone moiety is important for eliciting potent ACAT inhibition.

Chemical modification and structure-activity relationships of pyripyropenes. 2. 1,11-Cyclic analogs.

A series of 1,11-cyclic analogs of pyripyropene A were prepared. Replacement of the 1,11-acyl groups of pyripyropenes with 1,11-cyclic acetals effectively improved in vitro acyl CoA:cholesterol acyltransferase (ACAT) inhibitory activity. Especially noteworthy is benzylidene acetal analog 35, the most potent inhibitor (IC50 = 5.6 nM) among the derivatives prepared so far, which showed 16 times more potent inhibitory activity than pyripyropene A.

Chemical modification and structure-activity relationships of pyripyropenes. 1. Modification at the four hydroxyl groups.

Four hydroxyl groups of pyripyropenes have been modified and evaluated for their ability to inhibit microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity in vitro and to lower cholesterol absorption in vivo in a cholesterol-fed hamster. 7-O-n-Valeryl derivative (8c) improved the in vitro ACAT inhibitory activity (IC50 = 13 nM) about 7 times better than pyripyropene A. Introduction of methanesulfonyl group at 11-hydroxyl group (17a) increased both in vitro activity (IC50 = 19 nM) and in vivo efficacy (ED50 = 10 mg/kg).

Sequence analysis of the conserved protamine gene cluster shows that it contains a fourth expressed gene.

Structural data are presented on the protamine gene cluster (PGC) of human, mouse, rat, and bull. By restriction mapping we demonstrate that the organization of the protamine cluster is conserved throughout all four species, i.e., the genes are situated in a head to tail arrangement in the order: protamine 1-protamine 2-transition protein 2. Further, we established the nucleotide sequence of the entire human PGC (25 kb in total) and the 3' portion of the rat protamine cluster (PRM2 and TNP2 genes and intergenic region). In addition, a 1 kb fragment of the bovine and murine protamine cluster, situated between PRM2 and TNP2, was sequenced. This fragment is conserved regarding sequence, position, and orientation in all species examined, and was classified as likely coding region by gene recognition program GRAIL. Using the rat fragment as a probe in RNA blots, we detected a testis-specific signal of about 0.5 kb. Finally, we demonstrate a high density of Alu elements, both full and fragmented copies, in the human PGC and discuss their localization with respect to evolutionary and functional aspects.