Surgical program for craniosynostosis using three-dimensional solid model, combined with autologous blood transfusion in a Japanese craniofacial unit.

Since April 1997 at St. Mary's Craniofacial Unit, simulated surgery using a three-dimensional solid model made preoperatively was carried out, which enable sufficient autologous blood banking before the surgery. This study was conducted in eight patients presenting with plagiocephaly, brachycephaly, and oxycephaly consisting of simple craniosynostosis and syndromic craniosynostosis. Four cases (numbers 1-4) conducted with simulated surgery using three-dimensional solid models and fronto-orbital advancement with reshaping, as well as autologous blood transfusion were selected for the later-term group. For the first-term group, four cases (numbers 5-8) that were performed before the introduction of the abovementioned treatment were selected. The later-term group compared with the first-term group showed a decrease in operating time by an average of 1 hour, blood loss during surgery by 90 ml, and one fifth the amount of homologous blood transfusion. Also, 2 cases in the later-term group (cases 3 and 4) were able to avoid homologous blood transfusion, and they had autologous blood transfusion of 30 to 33 ml/kg. The surgical techniques will be improved with repeated cases of simulated surgery, aiming for the minimum invasive surgery. Also, the use of autologous blood transfusion is expected to increase as a less invasive surgery.

rDrak1, a novel kinase related to apoptosis, is strongly expressed in active osteoclasts and induces apoptosis.

This is the first report of a novel serine/threonine kinase, rabbit death-associated protein (DAP) kinase-related apoptosis-inducing protein kinase 1 (rDRAK1), involved in osteoclast apoptosis. We searched for osteoclast-specific genes from a cDNA library of highly enriched rabbit osteoclasts cultured on ivory. One of the cloned genes has a high homology with human DRAK1 (hDRAK1), which belongs to the DAP kinase subfamily of serine/threonine kinases. By screening a rabbit osteoclast cDNA library and 5'-RACE (rapid amplification of cDNA ends), we obtained a full length of this cDNA, termed rDRAK1. The sequencing data indicated that rDRAK1 has 88.0, 44.6, 38.7, and 42.3% identity with hDRAK1, DAP kinase, DRP-1, and ZIP (zipper-interacting protein) kinase, respectively. To clarify the role of DRAK1 in osteoclasts, we examined the effect of three osteoclast survival factors (interleukin-1, macrophage colony-stimulating factor, and osteoclast differentiation-inducing factor) on rDRAK1 mRNA expression and the effect of rDRAK1 overexpression on osteoclast apoptosis. The results suggested that these three survival factors were proved to inhibit rDRAK1 expression in rabbit osteoclasts. After transfection of a rDRAK1 expression vector into cultured osteoclasts, overexpressed rDRAK1 was localized exclusively to the nuclei and induced apoptosis. Hence, rDRAK1 may play an important role in the core apoptosis program in osteoclast.

Alcohol absorption inhibitors from bay leaf (Laurus nobilis): structure-requirements of sesquiterpenes for the activity.

Through a bioassay-guided separation using inhibitory activity on blood ethanol elevation in oral ethanol-loaded rat, various sesquiterpenes having an alpha-methylene-gamma-butyrolactone moiety, costunolide (1), dehydrocostus lactone (2), zaluzanin D (3), reynosin (4), santamarine (5), 3alpha-acetoxyeudesma-1,4(15),11(13)-trien-12,6alpha-+ ++olide (6) and 3-oxoeudesma-1,4,11(13)-trien-12,6alpha-olide (7), were isolated as the active principle from the leaves of Laurus nobilis (bay leaf, laurel). In order to characterize the structure requirement for the activity, several reduction products (2a-2d) and amino acid adducts (2e, 2f) of the alpha-methylene-gamma-butyrolactone moiety were synthesized from 2 and the inhibitory activities of these sesquiterpenes, together with alpha-methylene-gamma-butyrolactone (12) and its related compounds (13-16), were examined. These results indicated that the gamma-butyrolactone or gamma-butyrolactol moiety having alpha-methylene or alpha-methyl group was essential for the inhibitory activity on ethanol absorption. Since 1, 2 and 12 showed no significant effect on glucose absorption, these sesquiterpenes appeared to selectively inhibit ethanol absorption. In addition, the acute toxicities of 1 and 2 in a single oral administration were found to be lower than that of 12.

Medicinal foodstuffs. XVIII. Phytoestrogens from the aerial part of Petroselinum crispum MIll. (Parsley) and structures of 6"-acetylapiin and a new monoterpene glycoside, petroside.

In the course of our screening for natural estrogenic compounds from Occidental medicinal herbs, the extracts of several herbs were found to show proliferative activity in MCF-7 (an estrogen-sensitive breast cancer cell line). Among these active herbs, the methanolic extract from the aerial parts of Petroselinum crispum (parsley) showed potent estrogenic activity, which was equal to that of isoflavone glycosides from soybean. Through bioassay-guided separation, we isolated several flavone glycosides and a new flavone glycoside, 6"-acetylapiin, with estrogenic activity together with a new monoterpene glucoside, petroside. The structures of 6"-acetylapiin and petroside were characterized by the chemical and physicochemical evidence. Estrogenic activities of these flavone glycosides were found to be enhanced by removal of their glycoside moieties. The EC50 values (concentration needed to enhance the MCF-7 proliferation 50% compared to non-estrogen treated cell) of their aglycones are as follows, apigenin (1.0 microM), diosmetin (2.9 microM), and kaempferol (0.56 microM). The estrogenic activities of these flavones are nearly equal to those of the isoflavones, daidzein (0.61 microM) and genistein (0.60 microM). The methanolic extract of parsley, apiin, and apigenin restored the uterus weight in ovariectomized mice when orally administered for consecutive 7 days.

Effects of naloxone on the serum luteinizing hormone level and the number of Fos-positive gonadotropin-releasing hormone neurons in immature female rats.

To examine developmental changes in the number of gonadotropin-releasing hormone (GnRH) neurons activated by an opioid receptor antagonist in female rats, blood sampling and double-labeled immunocytochemistry for Fos and GnRH were performed after the injection of naloxone (NAL) in immature (postnatal d16 and d30) and mature female rats. Three age groups of rats were perfused with 4% paraformaldehyde-PB 90 min after the subcutaneous injection of NAL (2.5 mg/kg) or saline. All tissue incubation and staining for double-labeled immunocytochemistry were simultaneously performed. Although no significant developmental change was observed in the total number of GnRH neurons (p0.05), NAL-induced increases in serum luteinizing hormone (LH) concentrations were much greater in the d16 group than those in the d30 and mature groups (p<0.01). Conversely, Fos-positive GnRH neurons were rarely observed in d16, and some Fos-positive GnRH neurons were observed in the d30 group (p<0.05 vs. saline) and the mature group (p<0.01 vs. saline). These results suggest that opiatergic inhibitory system on GnRH neuron in immature female rats is different from that in mature female rats.

Chemical constituents from the leaves of Hydrangea macrophylla var. thunbergii (III): Absolute stereostructures of hydramacrosides A and B, secoiridoid glucoside complexes with inhibitory activity on histamine release.

Following the characterization of dihydroisocoumarin constituents, two secoiridoid glucoside complexes, called hydramacrosides A and B, were isolated from the leaves of Hydrangea macrophylla Seringe var. thunbergii Makino. The absolute stereostructures of hydramacrosides A and B were elucidated on the basis of chemical and physicochemical evidence, which included the application of the 13C-NMR glycosylation shift rule of 1,1'-disaccharides and the modified Mosher's method. Hydramacrosides A and B exhibited an inhibitory effect on histamine release from rat mast cells induced by an antigen-antibody reaction.

Classification of hypothalamic progestin-nonresponsive amenorrhea by secretion pattern of serum LH.

The pathogenesis of hypothalamic progestin-nonresponsive amenorrhea is unclear and this disease often fails to respond to treatment. The pulsatile patterns of diurnal and nocturnal secretion of serum LH as well as serum levels of melatonin were examined to improve the understanding of the pathogenesis and to develop strategies for the management of a severe type of hypothalamic amenorrhea. Four types of LH pulsatile patterns were observed: a) no pulse during the day or night (Group 1); b) more than 1 pulse only at night (Group 2); c) only 1 pulse during the day and more than 2 pulses at night (Group 3); and d) more than 2 pulses during the day and at night (Group 4). Serum estradiol was less than 30 pg/mL, and the serum PRL and PRL response to TRH did not differ among the four groups. The basal level and the pulse amplitude of LH increased successively from Group 1 to Group 4. The serum level of melatonin at night was noticeably increased in Group 1 and correlated negatively with the LH pulse frequency at night. After 6-month hormone replacement therapy with estrogen and progesterone, the rate of improvement in ovarian function were 0%, 33.3%, 57.1% and 67.0% in Groups 1, 2, 3, and 4, respectively. In 5 patients, the LH pulse pattern was re-examined at 6 months, the LH pulsatile pattern was changed from that of Group 1 to that of Group 4, with a decrease in serum concentrations of nocturnal melatonin, indicating improved ovarian function. In conclusion, classification of patients according to the LH secretion pattern is useful in establishing the severity of hypothalamic disturbance in hypothalamic progestin-nonresponsive amenorrhea and in predicting its prognosis; in addition nocturnal melatonin can be used as a marker for severer cases of hypothalamic amenorrhea.

Effects of noradrenaline on GnRH-secreting immortalized hypothalamic (GT1-7) neurons.

Noradrenaline (NA) is one of the most important neurotransmitters involved in the regulation of gonadotropin-releasing hormone (GnRH) secretion. In this study, the effects of NA on GnRH secretion, intracellular Ca2+ concentrations ([Ca2+]i), and membrane potentials were investigated in immortalized hypothalamic neurons (GT1-7) to determine the direct effects of NA on GnRH cells. Cells were perfused in a plastic minicolumn, and GnRH concentrations of the effluents were measured. NA increased the release of GnRH in a dose-dependent manner. Cells were loaded with a 4 microM Fura 2-AM, and the ratio of the intensities of fluorescent emission at 510 nm with excitation at 340 and 380 nm was calculated at 100-ms intervals. NA increased the [Ca2+]i responses of single GnRH cells dose-dependently. The NA-induced [Ca2+]i increase was attenuated in the absence of extracellular calcium and was blocked by the beta-adrenergic antagonist propranolol, but not by the alpha-adrenergic antagonist phentolamine. The cell membrane potential was recorded with a whole-cell patch clamp amplifier with glass-electrodes. NA induced membrane depolarization under current-clamp conditions. The depolarization was also inhibited by propranolol, but not by phentolamine. The results show that NA directly affects the membrane potential of GT1-7 cells via beta-adrenergic receptors and induces Ca2+ mobilization; these effects stimulate GnRH secretion.

Neuronal synchronization and ionic mechanisms for propagation of excitation in the functional network of immortalized GT1-7 neurons: optical imaging with a voltage-sensitive dye.

Immortalized gonadotropin releasing hormone (GnRH) neurons (GT1 cell line) in culture release GnRH in a pulsatile manner, suggesting that GT1 cells form a functional neuronal network. Optical imaging techniques and a voltage-sensitive fluorescent dye (RH795) were used to study the mechanism of neuronal synchronization and intercellular communication in cultured GT1-7 cells (one of the subclones of the GT1 cell line). The majority (79%) of GT1-7 cells in contact with one another revealed synchronized fluctuations in spontaneous neuronal activity. When a cell in contact with other cells was electrically stimulated, the evoked excitation was propagated to neighbouring cells. The ionic mechanisms involved in the propagation of electrical signals between interconnected GT1-7 cells were investigated using various blockers of Na+, Ca2+ and K+ channels. The propagation of stimulus-evoked excitation was prevented by the voltage-dependent Na+ channel blocker tetrodotoxin. It was also prevented by the voltage-dependent Ca2+ channel blockers, Ni+ (nonselective), nimodipine (L-type) and flunarizine (T-type > L-type), but not apparently affected by omega-agatoxin IVA (P- and Q-type) and omega-conotoxin MVIIA (N-type). The propagation was not influenced by the K+ channel blockers, quinine, tetraethylammonium and Ba2+, but in some cases, it was enhanced by 4-aminopyridine (4-AP) and prevented by apamin. These results suggest that voltage-dependent Na+ channels and L- and T-type Ca2+ channels are involved in the propagation of electrical signals in the GT1-7 neuronal network. Ionic mechanisms, through 4-AP- or apamin-sensitive K+ channels, also seem to be involved in the regulation of signal propagation. These mechanisms may underlie the functioning of the neuronal network formed by immortalized GnRH neurons.

[Antimicrobial and anti-inflammatory actions of tea-leaf saponin].

Antimicrobial and anti-inflammatory actions of tea-leaf saponin, which was a mixture of saponin separated from leaves of Camellia sinensis var. sinensis, were investigated. Tea-leaf saponin showed relatively high antimicrobial activity against pathogenic dermal fungi and its MIC value for Microsporum audouinii was 10 microgam/ml. On the other hand, tea-leaf saponin inhibited rat paw edema induced by carrageenin in a dose dependent manner. Activation of hyaluronidase, one of the enzymes involved in inflammatory reactions, was inhibited by tea-leaf saponin. It was also found that tea-leaf saponin antagonized the action of leukotrien D4, one of the chemical mediators of inflammatory reactions. Any symptom of toxic reaction was not observed when tea-leaf saponin was administered orally to mice at a dose of 2000 mg/kg.

A new glucuronide saponin from tea leaves (Camellia sinensis var. sinensis.

A new glucuronide saponin (1) was isolated as its methyl ester (2) from the leaves of Camellia sinensis var. sinensis. On the basis of its spectral data and the results of chemical degradation, the structure was elucidated to be 3-O-[beta-D-galactopyranosyl(1-->2)-[beta-D- xylopyranosyl(1-->2)-alpha-L-arabinopyranosyl(1-->3)]- beta-D-glucuronopyranosyl]-21-O-cinnamoyl-16,22-di-O-acetylbarr ingtogenol C.

Pupillary responses in normal subjects following auditory stimulation.

To clarify pupillary responses of humans following auditory stimuli, we studied both eyes of 61 normal subjects using a computed pupillograph. Unilateral auditory stimulation elicited pupillary dilatation in all cases. Pupillary responses were classified according to duration as being either "long" or "short". The duration of dilatation was 1530 +/- 320 ms (mean +/- SD) in the long-lasting group (n = 45) and 850 +/- 250 ms in the short-lasting group (n = 16). The latency time for dilatation was 460 +/- 80 ms. Both eyes of each subject showed the same response. Two drops of 10% guanethidine, a sympathetic blocking agent, were applied to one eye of 3 subjects. Although the early phase of dilatation was barely affected, the late phase was inhibited, as seen in long-lasting dilatation. The short-lasting response was unaffected. We conclude that the long-lasting response consists of an early pupillary dilatation due to inhibition of parasympathetic nervous activity and a late dilatation due to excitation of sympathetic activity. The short-lasting response is produced only by inhibition of the parasympathetic component.

Segregation analysis of IgE responses to Cryptomeria japonica pollen antigen in vivo.

The IgE response to Cryptomeria japonica pollen antigen (CPAg) in vivo was determined by radioimmunoassay of the plasma of 525 members from 98 families with known nasal allergies. Based on responses, patients were classified into a non-responder or low-responder group (non/low) and a high-responder group. Segregation analysis revealed that the IgE non/low responsiveness to CPAg involved a single dominant trait. The gene frequency was calculated to be 0.44-0.60. The IgE non/low response to CPAg was found to be mediated by CPAg-specific suppressor T cells. These findings demonstrated that the phenotypic variation of IgE responsiveness to CPAg is not due the immune response gene, but rather is mediated by the immune suppression gene for CPAg, via CPAg-specific suppressor T cells.

Central electrophysiologic correlates of pulsatile luteinizing hormone secretion in the rhesus monkey.

Characteristic increases in neuronal activity coincident with the pulsatile release of luteinizing hormone from the pituitary gland have been recorded from electrodes chronically implanted in the medial basal hypothalamus of the rhesus monkey. This electrophysiologic manifestation of the hypothalamic 'pulse generator' which governs the secretion of hypothalamic luteinizing hormone releasing hormone provides, for the first time, direct access to the central component of the neuroendocrine control system which regulates reproductive processes in this higher primate.

Serum HDL-cholesterol-increasing and fatty liver-improving actions of Panax ginseng in high cholesterol diet-fed rats with clinical effect on hyperlipidemia in man.

Effects of Panax ginseng on plasma and hepatic lipids were investigated in the high cholesterol diet-fed rats and in patients with hyperlipidemia. Oral administration of red ginseng powder reduced plasma total cholesterol, triglyceride and NEFA, while plasma HDL-cholesterol was elevated. Platelet adhesiveness was also reduced by ginseng administration. The plasma lipid-improving actions were also observed in patients with hyperlipidemia. Hepatic cholesterol and triglyceride contents were decreased and phospholipid increased by ginseng administration in the high cholesterol diet-fed rats, corresponding to improvement of the fatty liver.