Multicenter observational study comparing sedation/analgesia protocols for laser photocoagulation treatment of retinopathy of prematurity.

OBJECTIVE: The aim of this study was to identify the best sedation/analgesia protocol for laser photocoagulation (PC) of retinopathy of prematurity (ROP). STUDY DESIGN: This multicenter observational study included five hospitals, each using a specific sedation/analgesia protocol: local anesthesia with oxybuprocaine hydrochloride (Group L); intravenous pentazocine (Group P); intravenous fentanyl (Group F); air, oxygen and sevoflurane (AOS) inhalation (Group I). The groups were compared for pain responses, vital signs and adverse events. RESULTS: Heart rates and systemic blood pressures were elevated by PC in Groups L and P and Groups L, P and F, respectively. Moreover, poor analgesic efficacy was recognized in Groups L, P and F. In contrast, Group I experienced hypothermia, enteral feeding intolerance and apnea more frequently. CONCLUSION: From the viewpoint of sedation/pain relief, AOS anesthesia should be the best protocol. However, considering all the various factors together, the most reasonable one can be varied based on the patient's condition and hospital.

Identification of novel hypocholesterolemic peptides derived from bovine milk beta-lactoglobulin.

This study was designed to clarify the mechanisms of hypocholesterolemic action of beta-lactoglobuline tryptic hydrolysate (LTH) and to identify the novel hypocholesterolemic peptide derived from LTH by screening using Caco-2 cells and animal studies. Serum and liver cholesterol levels were significantly lower in rats fed LTH than in those fed casein tryptic hydrolysate (CTH). The present study suggests that the inhibition of micellar solubility of cholesterol which causes the suppression of cholesterol absorption by a direct interaction between cholesterol mixed micelles, and LTH in the jejunal epithelia is part of the mechanism underlying the hypocholesterolemic action of LTH. Though no one could trace the hypocholesterolemic peptide to any protein origin, we identified, for the first time, a novel hypocholesterolemic peptide, Ile-Ile-Ala-Glu-Lys (IIAEK). Surprisingly, the present study provides the first direct evidence that a new hypocholesterolemic peptide derived from beta-lactoglobuline can powerfully influence serum cholesterol levels and exhibit a greater hypocholesterolemic activity in comparison with that of medicine, beta-sitosterol, in animal studies.

Interactions of a didomain fragment of the Drosophila sex-lethal protein with single-stranded uridine-rich oligoribonucleotides derived from the transformer and Sex-lethal messenger RNA precursors: NMR with residue-selective [5-2H]uridine substitutions.

Proteins that contain two or more copies of the RNA-binding domain [ribonucleoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to be involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, residue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the present study, we analyzed the interaction of a Drosophila Sex-lethal (Sx1) protein fragment, consisting of two RBDs (RBD1-RBD2), with two distinct target RNAs derived from the tra and Sxl mRNA precursors with guanosine and adenosine, respectively, in a position near the 5'-terminus of a uridine stretch. First, we prepared a [5-2H]uridine phosphoramidite, and synthesized a series of 2H-labeled RNAs, in which all of the uridine residues except one were replaced by [5-2H]uridine in the target sequence, GU8C. By observing the H5-H6 TOCSY cross peaks of the series of 2H-labeled RNAs complexed with the Sx1 RBDI-RBD2, all of the base H5-H6 proton resonances of the target RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other target RNAs, GU2GU8, AU8, and UAU8, were assigned by comparison with those of GU8C. We found that the uridine residue prior to the G or A residue is essential for proper interaction with the protein, and that the interaction is tighter for A than for G. Moreover, the H1' resonance assignments were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the unusual C2'-endo ribose conformation in the complex.

Human natural tumor necrosis factor alpha induces multiple endocrine and hematologic disorders in rats.

Slc:Wistar male rats treated with human natural tumor necrosis factor alpha (hn TNF-alpha, 3 X 10(5) Japan reference units/kg intravenously) for 3 months showed histologic vacuolation of basophils in the anterior pituitary, hyperplasia of the thyroidal follicular epithelium, and hyperplasia of the testicular interstitial cells. The vacuolated basophils were immunohistochemically shown to be thyrotrophs. In addition, there were decreases in plasma levels of triiodothyronine (T3), thyroxin (T4), and testosterone, and an increase in thyroid-stimulating hormone (TSH). The number of lymphocytes in the marginal zones of lymphoid follicles in spleen and lymph nodes and B-lymphocytes in the peripheral blood decreased. Hyperplasia of hematopoietic cells in the bone marrow and decreases in both leukocytes and erythrocytes in the peripheral blood were prominent. Hyperplasia of bile ductular epithelial cells with periportal mononuclear cell infiltration in the liver and increased cellularity in alveolar walls in the lung were also characteristic. In in vitro studies, hn TNF-alpha inhibited both proliferation and peroxidase activity of thyroid follicular epithelial cells. These findings demonstrate that hn TNF-alpha may induce histologic vacuolation of thyrotrophs by causing a decrease in plasma levels of T3 and T4; hyperplasia of the thyroid follicular epithelium, which may be attributed to the increased plasma level of TSH; hyperplasia of testicular interstitial cells, by lowering the plasma level of testosterone; hyperplasia of bile ductular epithelial cells; hyperplasia of hematopoietic cells in bone marrow; and the increase in cellularity in pulmonary alveolar walls. In addition, hn TNF-alpha may suppress the differentiation of B-lymphocytes.

Protection against preterm delivery in mice by urinary trypsin inhibitor.

OBJECTIVE: To investigate the precise mechanism by which urinary trypsin inhibitor suppresses cytokine production in the prevention of preterm delivery. METHODS: In vivo and in vitro studies were performed using ascites and peritoneal macrophages obtained on day 15 of pregnancy from female C3H/HeN mice that had been impregnated by B6D2F1 male mice. Lipopolysaccharide receptor, the intracellular signal transduction system, and nuclear factor-kappaB level were examined. RESULTS: In the in vivo study, we found that urinary trypsin inhibitor ameliorated the deterioration of intraperitoneal conditions induced by lipopolysaccharide (ie, increases in ascitic volume, peritoneal cell count, and tumor necrosis factor-alpha level) and caused a decrease in the binding of lipopolysaccharide to mouse macrophages. In the in vitro studies, urinary trypsin inhibitor decreased the binding capacity of lipopolysaccharide for its receptor, blocked the intracellular signal transduction induced by lipopolysaccharide, and decreased the nuclear factor-kappaB level. Increases were induced in the binding capacity of the macrophages for urinary trypsin inhibitor and its incorporation into them in the presence of lipopolysaccharide. CONCLUSION: We postulate that urinary trypsin inhibitor may suppress the production of inflammatory cytokines induced by lipopolysaccharide in mouse peritoneal macrophages through suppression of the lipopolysaccharide receptor, inhibition of the intracellular signal transduction system, and decrease in the nuclear factor-kappaB level.

Lipoteichoic acid induces preterm delivery in mice.

The purpose of this study was to determine whether or not lipoteichoic acid (LTA) could induce preterm delivery in mice. On days 15 and 17 of pregnancy, female C3H/HeN mice impregnated by male B6D2F1 mice were given intraperitoneal injections of LTA (12.5-75 mg/kg, single dose or repeated doses at a 3-h interval). We examined the changes in cervix, placental trophoblasts, and plasma and amniotic fluid concentrations of interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) after dosing with LTA. In addition, the effect of LTA on the contraction of isolated uterine muscle from pregnant mice was also measured. The incidence of preterm delivery was highest (100%), when the pregnant animals were treated with 75 mg/kg LTA twice on day 15 of pregnancy or with 25 mg/kg LTA twice on day 17 of pregnancy. LTA-accelerated cervical ripening and placental abruption preceding the onset of preterm delivery, as well as increased plasma and amniotic fluid concentrations of IL-1alpha, IL-6, and TNF-alpha. Also, LTA increased contraction of uterine muscle strips. In conclusion, LTA induced preterm delivery in mice in the same manner as lipopolysaccharide (LPS), but the effective dose of LTA was larger than that of LPS.

Tocolytic effect of magnesium sulfate and/or urinary trypsin inhibitor against lipopolysaccharide-induced preterm delivery in mice.

BACKGROUND: The purpose of this investigation was to evaluate the effect of magnesium sulfate (magnesium) alone or along with urinary trypsin inhibitor (UTI) in a mouse model in the prevention of preterm delivery. METHODS: On day 17 of pregnancy, female C3H/HeN mice impregnated by male B6D2F1 mice were given two intraperitoneal injections of lipopolysaccharide (LPS; 50 microg/kg) at a 3-hour interval, which treatment induced a 100% incidence of preterm delivery within 25 hours of the second dose. Magnesium (4, 5, 6, 7, or 8 mg/hr, s.c.), UTI (25 x 10(4) units/kg, i.p.), magnesium (5 mg/hr, s.c.) plus UTI (25 x 10(4) units/kg, i.p.), saline solution (0.3 ml/hr, s.c.), or saline solution (0.1 ml/hr, s.c. and 2.5 ml/kg, i.p.) was administered to pregnant animals on day 18 of gestation. UTI was intraperitoneally given 5 times at 2-hour intervals from 8:00 am to 4:00 pm, and magnesium was infused subcutaneously and constantly from 8:00 am to 6:00 pm. In addition, the preventive effect of magnesium on LPS-induced contraction of uterine muscle strips and that of magnesium, UTI, or magnesium plus UTI on LPS-induced calcium influx in uterine smooth muscle cells were examined using muscle tissue isolated from pregnant mice on day 17 of gestation. RESULTS: The incidence of preterm delivery decreased significantly in a dose-dependent fashion with magnesium treatment, and there was a significant and synergistic decrease after combined treatment with magnesium plus UTI. The in vitro uterine contraction and calcium influx induced by LPS were significantly suppressed by magnesium. The latter was significantly suppressed by UTI and additively reduced by magnesium plus UTI. CONCLUSIONS: Combination therapy with magnesium plus UTI may possibly be helpful for preventing preterm delivery in humans without the severe side effects induced by hypermagnesemia.

An RNA aptamer to the xanthine/guanine base with a distinctive mode of purine recognition.

RNAs that bind to xanthine (2,6-dioxypurine) were isolated from a population of 10(12) random sequences by in vitro selection. These xanthine-binding RNAs were found to have a 10 nt consensus sequence at an internal loop in the most probable secondary structure. By trimming one of the xanthine-binding RNAs, a representative xanthine-binding RNA (designated as XBA) of 32 nt residues was prepared. The dissociation constant of this RNA for xanthine was determined to be 3.3 microM by equilibrium filtration experiments. The XBA RNA can bind to guanine as well, whereas it hardly accommodates adenine, cytosine or uracil. The K d values for various xanthine/guanine analogues were determined, and revealed that the N1H, N7 and O6 moieties of the ligand are involved in the binding with the XBA RNA. The ribonuclease sensitivities of some internal-loop residues changed upon the addition of xanthine, suggesting that the internal loop of the XBA RNA is involved in the ligand binding. Interestingly, the consensus sequence of the xanthine/guanine-binding RNAs is the same as a sequence in one of the internal loops of the hairpin ribozyme, except for a substitution that is neutral with respect to xanthine/guanine binding.

Induction of Leydig cell tumors by lacidipine via up-regulation of the LHRH receptor on Leydig cells in rats.

Long-term (78 weeks) administration of lacidipine, a dihydropyridine calcium antagonist, increased the incidence of Leydig cell tumors (LCTs) in Sprague-Dawley rats. Lacidipine also increased and decreased the plasma luteinizing hormone (LH) and testosterone levels, respectively. Leydig cells from lacidipine-treated rats showed increases in luteinizing hormone-releasing hormone (LHRH) receptor expression, protein kinase C (PKC) activity, expression of proto-oncogenes, and 5-bromodeoxyridine uptake; whereas their calcium level, LH receptors, and testosterone content decreased. These data suggest that LHRH receptors play an important role in the development of rat LCTs induced by lacidipine, which activates a cascade of cell cycle-regulatory genes via PKC. When isolated Leydig cells were cultured with lacidipine or nicardipine, these changes in rat Leydig cells were not demonstrable in mice and monkeys, species having many fewer testicular LHRH receptors than rats. Thus, lacidipine may pharmacologically induce LCTs in rats but not in mice, with the difference depending on the presence or absence of testicular LHRH receptors. The induction of LCTs by lacidipine in rats is unlikely to occur in humans, since their Leydig cells lack LHRH receptors.

Effects of dienogest, a synthetic steroid, on experimental endometriosis in rats.

OBJECTIVE: Dienogest, a synthetic steroid with progestational activity, is used as a component of oral contraceptives and is currently being evaluated clinically for the treatment of endometriosis. The present study was conducted to confirm the effects of dienogest on experimental endometriosis in rats and to elucidate its mechanism of action. DESIGN: Experimental endometriosis induced by autotransplantation of endometrium in rats. METHODS: Endometrial implants, immune system, and bone mineral were investigated after 3 weeks of medication. RESULTS: Dienogest (0.1-1 mg/kg per day, p.o.) reduced the endometrial implant volume to the same extent as danazol (100 mg/kg per day, p.o.). Simultaneously, dienogest ameliorated the endometrial implant-induced alterations of the immune system: i.e. it increased the natural killer activity of peritoneal fluid cells and splenic cells, decreased the number of peritoneal fluid cells, and decreased interleukin-1beta production by peritoneal macrophages. In contrast, danazol (100 mg/kg per day, p.o.) and buserelin (30 microg/kg per day, s.c.) had none of these immunologic effects. Additionally, combined administration of dienogest (0.1 mg/kg per day) plus buserelin (0.3 microg/kg per day) suppressed the bone mineral loss induced by buserelin alone, with no reduction of the effect on endometrial implants. In vitro studies on dienogest revealed an antiproliferative effect on rat endometrial cells due to inhibition of protein kinase C activity plus a partial progestational effect. CONCLUSIONS: Dienogest appears to be a potent agent with mechanisms of action different from those of danazol and GnRH agonists currently available for the treatment of endometriosis.

Trophoblastic apoptosis in mice with preterm delivery and its suppression by urinary trypsin inhibitor.

OBJECTIVE: To investigate histopathologic changes of the placenta in mice with preterm delivery induced by lipopolysaccharide and the effect of urinary trypsin inhibitor. METHODS: Female C3H/HeN mice impregnated by male B6D2F1 mice were treated with lipopolysaccharide (50 micrograms/kg, intraperitoneally) or lipopolysaccharide plus urinary trypsin inhibitor (25 x 10(4) U/kg, intraperitoneally). At 3, 6, 9, and 18 hours after the second dose of lipopolysaccharide, and at delivery in the control and urinary trypsin inhibitor-treated groups, the concentrations, of interleukin-1 alpha and tumor necrosis factor-alpha were determined in serum and amniotic fluid. Subsequently, the placentas were examined. In the same manner, we examined mice treated with interleukin-1 alpha (250 micrograms/kg, subcutaneously) on day 15 of pregnancy and intact mice on days 15 and 18 of pregnancy as well as at delivery. To assess the direct action of cytokines, we cultured placental slices with tumor necrosis factor-alpha, interleukin-1 alpha, or tumor necrosis factor-alpha plus urinary trypsin inhibitor and examined them morphologically. RESULTS: Control mice were characterized by trophoblastic apoptosis and increased serum levels of tumor necrosis factor-alpha and interleukin-1 alpha. In contrast, urinary trypsin inhibitor-treated mice showed suppression of apoptosis and lower cytokine levels. Interleukin-1 alpha induced trophoblastic apoptosis and increased the serum level of tumor necrosis factor-alpha. The in vitro study showed that tumor necrosis factor-alpha directly induced trophoblastic apoptosis in placental slices. CONCLUSION: We demonstrated that trophoblastic apoptosis occurs in the placentas of a mouse model with preterm delivery induced by lipopolysaccharide. We postulated that apoptosis may lead to placental abruption, and its development may be prevented by treatment with urinary trypsin inhibitor.

Amiodarone induces two different types of disorders in mouse alveolar macrophages.

It has been reported that amiodarone induces disorders of alveolar macrophages and pulmonary fibrosis, but the mechanism is not well-understood. This study was performed to elucidate the toxic mechanism from the standpoint of cellular function. Using alveolar macrophages obtained from a male Slc:ICR mouse, several injuries caused by amiodarone were compared to those caused by amantadine and mianserin as cationic amphiphilic drugs (CADs). As parameters for the drug effects, H(+)-ATPase and acid sphingomylinase activities, cellular pH, cytokine and prostaglandin releases, phagocytosis and neutral red uptake were measured. Amiodarone decreased H(+)-ATPase activity initially and subsequently increased cellular pH and decreased acid sphingomyelinase activity. These changes, which were also observed with amantadine and mianserin, were considered to be CAD-related. Amiodarone increased cytokine and prostaglandin releases and suppressed neutral red uptake and phagocytosis. These changes, being not induced by amantadine and mianserin, were considered to be specific for amiodarone. The above data suggest that amiodarone has two types of toxic effects on alveolar macrophages.

Toxicity of amiodarone on mouse pulmonary endothelial cells cultured with or without alveolar macrophages.

This study was designed to specify the toxicity of amiodarone toward mouse pulmonary endothelial cells in comparison with that of another cationic amphiphilic drug, i.e., mianserin. These examinations were performed in the absence and presence of mouse alveolar macrophages under transmembrane co-culture or in direct contact with the endothelial cells to assess the contribution of macrophages to the toxicities toward the endothelial cells. As a result of 24-hr treatment, amiodarone caused a decrease in cell viability, in H(+)-ATPase, acid sphingomyelinase, and acid phospholipase A2 activities, and in neutral red uptake, and an increase in permeability of the endothelial cells. Because the magnitude of changes in the endothelial cells was the greatest under direct contact with macrophages, and was the mildest without macrophages, macrophages were considered to enhance the toxicity of amiodarone toward the endothelial cells. Additionally, the toxic effect of amiodarone on the cells was depressed by pretreatment of them with docosahexaenoic acid (DHA) or alpha-tocopherol for 2 days and co-treatment with these agents for 1 day, but not with prednisolone or indomethacin co-treatment. DHA and alpha-tocopherol protected endothelial cells from the toxicity of amiodarone. The effect was more potent for DHA than alpha-tocopherol.

Role of urinary trypsin inhibitor in the maintenance of pregnancy in mice.

OBJECTIVE: To investigate the mechanisms whereby urinary trypsin inhibitor prevents lipopolysaccharide-induced preterm delivery in mice. METHODS: On day 15 of pregnancy, C3H/HeNCrg female mice impregnated by Crg:B6D2F1 male mice were treated intraperitoneally with lipopolysaccharide (50 micrograms/kg, twice at a 3-hour interval) to induce preterm delivery. Urinary trypsin inhibitor (2.5 x 10(4), 7.5 x 10(4), or 25 x 10(4) units/kg, ten times at 1-hour intervals) or saline solution was administered intraperitoneally to the animals. RESULTS: The incidence of preterm delivery was significantly decreased on a dose-related basis by urinary trypsin inhibitor treatment. Urinary trypsin inhibitor prevented the morphologic and functional changes in fetal membranes and cervical ripening preceding the onset of preterm delivery. Urinary trypsin inhibitor also suppressed the increase in plasma and amniotic fluid concentrations of interleukin-1 alpha, interleukin-6, and tumor necrosis factor-alpha after the lipopolysaccharide dosing in this animal model for preterm delivery. CONCLUSION: Urinary trypsin inhibitor prevents the pathogenicity of preterm delivery through the suppression of cytokine production.

Effect of amiodarone on cytokine release and on enzyme activities of mouse alveolar macrophages, bone marrow macrophages, and blood monocytes.

The effect off amiodarone, a cationic amphiphilic drug, on cytokine release from, and on protein kinase C (PKC) activity of, mouse alveolar macrophages, bone marrow macrophages, and blood monocytes was examined. In addition, its effect on three enzymes in these cells was also determined. Amiodarone suppressed the growth of all cell types at high doses. As regards cytokine release, amiodarone caused an increase in interleukin-1 (IL-1) alpha, IL-1 beta, and tumor necrosis factor (TNF) alpha release from alveolar macrophages but not from marrow macrophages and monocytes. PKC activity was increased by amiodarone only in alveolar macrophages. And the treatment with amiodarone severely suppressed the H(+)-ATPase, sphingomyelinase, and phospholipase A2 activities in alveolar macrophages. But these enzyme activities in bone marrow macrophages and monocytes were not suppressed so much as in alveolar macrophages. This current study indicated that mouse alveolar macrophages treated with amiodarone undergo suppression of H(+)-ATPase, resulting in suppression of sphingomyelinase and phospholipase A2 activity, and in activation of PKC activity and release of cytokines. It also showed that changes in activities of all three enzymes in alveolar macrophages are different from those in bone marrow macrophages and monocytes with respect to reactivity toward amiodarone.

Effect of amiodarone on release of cytokines from mouse alveolar macrophages pretreated with eicosapentaenoic acid.

I studied the effect of amiodarone, a cationic amphiphilic drug, on the cytokine release and protein kinase C (PKC) activity of mouse alveolar macrophages. In addition, I examined the relationship between amiodarone and eicosapentaenoic acid (EPA) with respect to the cytokine release. The decrease in cell number caused by amiodarone was depressed by pretreatment of the macrophages with EPA for 2 days and co-treatment for 1 day. These changes reflected the potency of EPA to protect against the cell injury elicited by amiodarone. As regards to the cytokine release, amiodarone caused an increase in interleukin (IL)-1 alpha, IL-1 beta and tumor necrosis factor (TNF) alpha release from macrophages. As EPA suppressed this increase in cytokine levels, I considered that the protective effect of EPA may be extended to the acute release of cytokines. PKC activity was increased by amiodarone, and this increase was depressed by EPA. These changes were well-related to the results on cytokine levels in this study, indicating that amiodarone firstly activated PKC, leading to the stimulation of release of cytokines and that pretreatment with EPA prevented these effects. I conclude that mouse alveolar macrophages treated with amiodarone show activated release of cytokines and that EPA depresses these increases, thereby demonstrating EPA's anti-inflammatory effect and protective action against injury of alveolar macrophages.

Cell counts in peripheral blood and bone marrow of male CBA/N mice.

Measurements for blood and bone marrow cell counts were carried out in male CBA/N mice aged 3, 6, 9, 12, and 26 weeks and the values were compared with those in normal mice. In the peripheral blood, CBA/N mice showed decreasing numbers of leukocytes with age, without marked changes in the differential cell count. In the bone marrow of CBA/N mice, the total number of femoral bone marrow cells was low in later life (26 weeks of age), and differential cell counts revealed a high granuloid/erythroid ratio at 3 weeks of age due to a low erythroblast count.

Effects of medroxyprogesterone acetate and beta-estradiol on interleukin-6 production from osteoblasts and bone marrow macrophages of Wistar rats of different ages.

Using 18-week-old and 52-week-old Wistar rats, we examined interleukin-6 (IL-6) production from osteoblasts and bone marrow macrophages treated with medroxyprogesterone acetate (MPA) and/or beta-estradiol. The level of IL-6 production by osteoblasts was increased by treatment with MPA and, reversely, decreased by treatment with beta-estradiol. These changes were especially remarkable in osteoblasts of 52-week-old rats. Additionally, and in contrast, the production of IL-6 by bone marrow macrophages was not significantly changed by treatment with both agents. These data suggest that because the increased production of IL-6 by osteoblasts treated with MPA in opposition with beta-estradiol, MPA should be careful for osteoporosis dependent upon osteoclasts activated by IL-6. Finally, there was a marked difference in the amount of IL-6 produced between osteoblasts and bone marrow macrophages.