The entrapment of kojic oleate in bilayer vesicles.

The entrapment of kojic acid and its newly synthesized ester (kojic oleate) has been evaluated. Kojic oleate was synthesized by DCC (N,N'-dicyclohexylcarbodiimide, DCC)/(4-(N,N-dimethylamino)pyridine, DMAP) esterification method and identified by FAB-MS and 1H NMR. The synthesized product was mainly 7-O-kojic oleate with more than 80% yield. It was entrapped in vesicular membrane prepared from 9.5:9.5:1.0 molar ratio of amphiphiles (Span 60, Tween 61 or DPPC), cholesterol and dicetyl phosphate. Kojic acid was encapsulated in the water compartment of these vesicles in order to confirm the vesicle formation. The morphology and particle size of the vesicles were characterized by an optical microscope and transmission electron microscope (TEM). The entrapment efficiencies of kojic acid and kojic oleate in the vesicles were investigated by dialysis and column chromatography, respectively. The contents of the entrapped kojic acid and kojic oleate were assayed by HPLC. The entrapment efficiency of kojic acid was 0.01-0.04 mol, whereas kojic oleate gave higher entrapment efficiency of 0.25-0.35 mol/mol of the total compositions of amphiphile/cholesterol/dicetyl phosphate. Structural modification of kojic acid improved its entrapment in the vesicles. Tween 61 vesicles could entrap kojic oleate more than did Span 60 vesicles. The pi-A isotherms revealed the lower area per molecule of Span 60, which formed a more rigid pack of its molecule on air/water interface than that of Tween 61. This implied the high rigidity of vesicular membrane prepared with Span 60 led to the lower amount of kojic oleate entrapped in the vesicles. From the release study of kojic acid through the dialysis membrane, it indicated that the intercalation of kojic oleate in the vesicular membranes did not significantly affect the release of kojic acid from the vesicles.

Expression pattern of a newt Notch homologue in regenerating newt retina.

We isolated part of a newt Notch homologue, N-Notch, from regenerating newt retina. The spatio-temporal pattern of N-Notch expression was studied by in situ hybridization at different stages of newt retinal regeneration. Proliferating cells were confirmed by the injection of bromodeoxyuridine (BrdU). In the early stage of regeneration, when the retina was one to two cells thick, all proliferating retinal progenitors expressed N-Notch. As the thickness of the retina increased with regeneration, N-Notch expression decreased in BrdU-positive cells on the vitreal side of the retina. Subsequently, presumptive retinal ganglion cells that were BrdU-negative cells appeared at the vitreal edge of the regenerating retina. These differentiating cells did not express N-Notch. Later, N-Notch expression decreased in the BrdU-positive cells on the scleral surface of the retina. Subsequently, presumptive photoreceptor cells that were BrdU-negative cells appeared in this region. These differentiating cells also did not express N-Notch. The proliferating retinal progenitors ceased expressing N-Notch and then stopped dividing during the differentiation of ganglion cells and photoreceptor cells. It was found that retinal regeneration involves the expression of an important developmental signaling molecule, Notch, in retinal progenitors and the expression of Notch ceased as cell differentiation proceeded during retinal regeneration.

The occurrence of apoptosis during retinal regeneration in adult newts.

The present study examined the occurrence of apoptosis, identified by an in situ technique for detecting DNA fragmentation, in the regenerating retina of adult newts following ablation of the retina. Apoptosis occurs in the initial phase of regeneration when retinal precursor cells are actively proliferating. In the late stage of regeneration, when two synaptic layers are forming, apoptosis occurs mainly in the ganglion cell layer and inner nuclear layer. We found that apoptosis occurred with proliferation, differentiation, formation of retinal layers and retinotectal projections during retinal regeneration. Our findings suggest that apoptosis is closely related to these phenomena.

Pax-6 expression during retinal regeneration in the adult newt.

The present study examined the expression of Pax-6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax-6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax-6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax-6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax-6 became similar to that of a normal retina. It was found that Pax-6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax-6 changed as cell differentiation proceeded during retinal regeneration.

Simultaneous measurement of spectroscopic and physiological signals from a planar bilayer system: detecting voltage-dependent movement of a membrane-incorporated peptide.

We developed an experimental system that can measure spectroscopic and physiological signals simultaneously from ion channels in a planar lipid bilayer, to study the relationship between the structure and function of the ion channels. While the membrane potential was clamped, fluorescent emission and ionic currents were measured simultaneously. The fluorescent emissions from a planar bilayer constructed in a specially designed chamber were monitored exclusively, and the signal intensity was measured with a photon-counting system. The intensity of fluorescence and spectral shape were measured successfully from the planar bilayer, with a high signal-to-noise ratio. The system can measure the intensity of fluorescence from a restricted area of the planar bilayer, with a diameter of 70 micrometer and a focal depth of 15 micrometer. The low background signal was achieved by optimizing the optical system. More than 95% of the measured fluorescence comes from the planar lipid bilayer. A 22-mer peptide with a sequence identical to that of the S4 segment of the electric eel sodium channel domain IV was synthesized and fluorescence-labeled. This peptide formed a voltage-dependent ion channel in a planar bilayer. The changes in the intensity of the fluorescence accompanying ionic currents generated by a voltage clamp suggest that voltage gating involves the insertion of the N-terminal of the peptide into the membrane. The electrical and optical signals were measured with a gate time of 10 ms. This measurement enabled the detection of movement of the membrane-incorporated peptides with channel opening.

TTX resistivity of Na+ channel in newt retinal neuron.

We examined voltage-dependent, TTX-resistant Na+ channels of newt retina (nRNaCh) electrophysiologically. IC50-TTX value of nRNaCh is more than 20 microM. We determined partial cDNA sequences of nRNaCh restricted to TTX binding sites (SS2 regions of all four repeats). While the amino acid sequences of SS2 regions of repeats II, III and IV of nRNaCh are identical to those of TTX-sensitive Na+ channels, only one amino acid in SS2 of repeat I of nRNaCh is different. nRNaCh have nonaromatic amino acid (Ala) in this site instead of the aromatic amino acid in a case of TTX-sensitive Na+ channels. Many studies suggested that the differences of TTX sensitivity of Na+ channels are decided by whether amino acid in this site is aromatic or not. Therefore nRNaCh acquire their TTX resistivity with the same mechanism as TTX-resistant cardiac Na+ channels do.

[Effect of posture strategies on preventing aspiration].

The purpose of this study was to determine the effect of postural change on preventing aspiration, and the types of swallowing disorder that were influenced by posture. Ninety-five patients that were examined by aspiration of barium during videofluoroscopic examination of oropharyngeal swallow assumed one of four postures (chin down, head rotation, chin down + head rotation, and head back to chin down). Postural strategies were successful in eliminating aspiration in 68(72%) of the 95 patients. Twenty five of the 68 patients, however, needed an additional change in bolus volume to eliminate aspiration. Effective postures eliminating aspiration were chin down for 43 patients, head rotation for 10 patients, chin down + head rotation for 12 patients, and head back to chin down for 3 patients. The successes rates of posture changes for each swallowing abnormality were 76% for reduced tongue base retraction, 74% for delay in triggering pharyngeal swallow, 74% for reduced laryngeal elevation, 73% for laryngeal paralysis, 68% for inefficient oral transit, and in 57% for cricopharyngeal dysfunction. Among individuals with cricopharyngeal dysfunction or with than three swallowing motility problems, the frequency of unsuccessful swallow despite postural change was significantly higher than that of successful swallow. These results indicated that the posture strategies in addition to control of bolus volume could prevent aspiration in more than 70% of patients, but the efficacy of posture techniques differed with the different types of swallowing disorder identified as causing the aspiration, and suggested that the posture strategies were less beneficial in individuals with cricopharyngeal dysfunction or exhibiting multiple swallowing disorders.

Cultured giant fiber lobe of squid expresses three distinct potassium channel activities in selective combinations.

Neurons from the giant fiber lobe (GFL) of squid Loligo bleekeri were dissociated and cultured. The ionic currents were recorded using whole-cell patch clamp methods. The sodium current and the noninactivating potassium current like those elicited by the giant axon were among the currents expressed in axonal bulbs and bulblike structures upon dissociation. Meanwhile axonless cell bodies did not elicit such currents. Axonless cell bodies and some bulblike structures elicited two kinds of inactivating potassium currents, the slow- and the fast-inactivating current, which differed in their inactivation kinetics and pharmacology. Within 24 hr of plating, the current composition remained the same. While the noninactivating current was not sensitive to 4-aminopyridine, the two inactivating currents were sensitive, the slow-inactivating current being more sensitive. Selective combinations of the sodium current and the three potassium currents expressed in different structures of the acutely dissociated GFL could have resulted from cellular control of synthesis and transportation of the channel proteins to the somatic and the axonal membrane. The sodium current and the noninactivating potassium current could be recorded from some axonless cell bodies maintained in culture for over three days, indicating that the separation of the giant axon from its somata could result in the transportation of the channels normally expressed on the giant axon membrane to the somatic membrane.

[Tonsillectomy in a boy liver transplant].

As of 1995, organ transplantation from cadaver donors is under debate in the Japanese Diet. Depending on what the Diet decides, more organ transplantations may be performed. Since cases of orthotopic liver transplantation (OLT) may increase, the need to perform surgical operations in post-OLT patients may arise. The purpose of this report is to enlighten Japanese otorhinolaryngologists on the post-transplant state. An 8-year-old boy who underwent OLT in Australia 7 years previously underwent successful tonsillectomy, adenoidectomy and insertion of ventilation tubes into both ears under general anesthesia (GA) to treat habitual angina, hypertrophy of the nasopharyngeal and palatine tonsils, and secretory otitis media. The optimal circumstances for operation require adequate but not excessive immunosuppression and a well-functioning graft. Vascular complications (VCs) such as hepatic artery thrombosis become rare after a few years post-OLT. However, once VCs occur the mortality rate of OLT patients is high, and excessive perioperative changes in circulation must be avoided. Immunosuppressive agents should be continued throughout the perioperative period and perioperative antibiotic prophylaxis should be employed, just as in non-transplant patients. Tonsillectomy is an effective means of prophylaxis for upper respiratory infection in habitual angina patients. Infection of an OLT patient may become critical because immunosuppressive agents to prevent rejection lower immune barriers and increase the risk of infection, and dose reduction may increase the risk of rejection. Tonsillectomy may also prevent a possible lympho-proliferative disorder (LPD). Tonsillar hypertrophy in OLT patients may be due to life-threatening LPD. Thus, tonsillectomy serves both as a prophylactic and curative measure against possible complications OLT may cause later, and therefore may improve the outcome of OLT.

Pigment granule migration and phototransduction are triggered by separate pathways in fly photoreceptor cells.

We studied the mechanism of pigment granule migration in fly photoreceptors. We found that these granules migrate transiently with red light stimulation. This suggests that excitation of metarhodopsin triggers granule migration. We also found that cyclic GMP is one of the activators of pigment granule migration. These findings suggest that pigment granule migration is triggered separately from phototransduction, which is initiated by the excitation of rhodopsin.

Mucopolysaccharidosis type IVA. N-acetylgalactosamine-6-sulfate sulfatase exonic point mutations in classical Morquio and mild cases.

Mucopolysaccharidosis type IVA (MPS IVA) results from a genetic deficiency of N-acetylgalactosamine-6-sulfate (Gal-NAc6S) sulfatase. We have identified two different exonic mutations causing GalNAc6S sulfatase deficiency in two unrelated Japanese families, in one patient with classical Morquio disease, and in two brothers with a mild form of MPS IVA. The nucleotide sequence of the full-length cDNA derived from a patient with classical Morquio disease revealed a two-base deletion at nucleotide position 1343-1344 (1344-1345 or 1345-1346) that altered the reading frame (designated 1342delCA). This mutation, inherited from the proband's consanguineous parents, was revealed by TaqI restriction analysis of a cDNA fragment amplified by the polymerase chain reaction. In the proband with the mild form of the disease, a C to G transversion at nucleotide 667 predicted the substitution of Lys for Asn204 (N204K). Since a new AluI site was created by the N204K mutation, restriction analysis indicated that the affected brothers were homozygous for this mutation, as confirmed by the finding that both their parents had this lesion. Transient expression in GalNAc6S sulfatase deficient fibroblasts of these two mutant alleles showed completely deficient or markedly decreased enzyme activities, thereby indicating that these two mutations were responsible for the enzyme deficiency.

An improved cryofixation method: cryoquenching of small tissue blocks during microwave irradiation.

The metal contact method of rapid freezing is greatly improved by irradiating the specimen with microwaves at 2.45 GHz for a short period of time (50 ms), while pushing the specimen onto the surface of the copper block cooled by liquid N2. The microwave irradiation, together with two technical improvements (a light-mass plunger and a recently developed beta-gel shock absorber) for preventing bounce, produces a good freezing zone for squid retina, with high reproducibility for each experimental trial, extending from the contact surface to a depth of about 15 microns, which is comparable to the depth obtained by the metal contact method using liquid He in the absence of microwave irradiation. A good freezing zone was also experimentally demonstrated in specimens of rat liver and heart muscle. Microwave irradiation does not have appreciable effects on the ultrastructure of squid retina. The mechanism underlying the improvement in the rapid freezing under the microwave irradiation is discussed.