Quantitative determination of lattice fluoride effects on the solubility and crystallinity of carbonated apatites with incorporated fluoride.

The purpose of this study was to evaluate quantitatively the effects of fluoride on the solubility and crystallinity of carbonated apatites (CAPs) after its incorporation into the crystal lattice using the metastable equilibrium solubility (MES) distribution method. Fluoride-incorporated CAPs (F-CAPs) of two different carbonate levels (3 and 5%) and fluoride contents from 0 to 20,000 µg/g were synthesized. X-ray diffraction experiments and Rietveld analysis were conducted to obtain crystallite microstrain and unit cell parameters. Acetate buffer MES solution media were prepared at two solution fluoride concentrations (0.2 and 2.0 mg/l) and at two pHs (5.0 and 5.7). The unit cell a-axis values of the F-CAPs were found to decrease as the fluoride content increased, consistent with the fluoride being incorporated into the crystal lattice. The fluoride concentrations in the MES solution media were high enough to provide a 'swamping' effect such that the fluoride released from the F-CAPs during dissolution was minimal in changing the solution fluoride concentration. Employing the MES distribution superposition method, it was shown that the surface complex possessing the fluorapatite (FAP) stoichiometry [Ca10(PO4)6F2] accounted for the MES distribution behavior of all experiments. In addition, the mean pIFAP [the value of -log(aCa(10)aPO4(6)aF(2)) calculated from the ionic activity product based on FAP stoichiometry of the MES dissolution media in which 50% of the F-CAPs had dissolved] correlated well with the crystallite microstrain parameters of the F-CAPs. The incorporated fluoride in the F-CAPs showed only modest effects on F-CAP crystallinity and solubility.

Cholesterol dissolution rate in micellar bile acid solutions: retarding effect of added lecithin.

In vitro studies on the dissolution rate of cholesterol monohydrate crystals in micellar bile acid solutions showed that the addition of lecithin decreases the dissolution rate even though lecithin increases the equilibrium solubility of cholesterol in these solutions. The reduction in rates caused by lecithin was attributed to a large crystal-solution interfacial barrier. An effective permeability coefficient for the interfacial barrier was calculated to be around 1.5 x 10(-5) centimeter per second for the transport of cholesterol molecules.

Bacillus cereus nosocomial infection from reused towels in Japan.

It was noticed that there was an increase in Bacillus cereus nosocomial infections in the summer from 2000 to 2005. In 2005, five bloodstream infections occurred in five patients related to catheter use. The causative strains were distinct from each other and belonged to novel multilocus sequence types (ST): ST365, ST366, ST367 and ST368. Two ST365 strains from two patients were further distinguished by pulsed-field gel electrophoresis. B. cereus contamination was observed with reused (dried and steamed) towels (>10(6)cfu/towel) and washing machines in hospital linen rooms. B. cereus strains from towels belonged to ST167, ST365, ST380 and ST382, and a proportion of these were the same, or similar, to strains from patients. All the hospital strains of B. cereus were distinct from those from food-poisoning strains (ST26, ST142, ST381). Ciprofloxacin resistance was observed only in hospital strains. Neither emetic toxin nor cytotoxin K gene, usually present in food poisoning strains, were found in the hospital strains, except for one patient isolate. The data suggest that specific B. cereus strains are circulating within a hospital, with genotypes, antibiotic susceptibilities and virulence gene patterns generally distinct from those of food poisoning, and that in Japan, towels are an important source of contamination, especially in summer.

Macrolide-lincosamide-streptogramin B resistance phenotypes and genotypes among Staphylococcus aureus clinical isolates in Japan.

In total, 269 methicillin-resistant Staphylococcus aureus (MRSA) and 434 methicillin-susceptible S. aureus (MSSA) isolates were investigated to determine their macrolide-lincosamide-streptogramin B (MLS(B)) resistance phenotypes and genotypes. The constitutive phenotype (61.3% in MRSA, 1.3% in MSSA) and erm(A) gene predominated among the 261 erythromycin-resistant MRSA isolates, while the inducible phenotype (38.7% in MRSA, 94.0% in MSSA) and erm(C) gene were more prevalent among the 150 erythromycin-resistant MSSA isolates. There was a higher incidence of the MLS(B) inducible phenotype compared with other countries, perhaps because MLS(B) antibiotics are not recommended as first-line agents against S. aureus in Japan.

Noninvasive measurement of phenylalanine by iontophoretic extraction in patients with phenylketonuria.

Phenylketonuria is an autosomal recessive disorder characterized by elevated concentrations of phenylalanine. Elevated phenylalanine concentrations can impair intellectual functions and the disease is treated with a lifelong diet and frequent monitoring of plasma phenylalanine concentrations. Previous in vitro studies have demonstrated the feasibility of iontophoretically enhanced transdermal transport of phenylalanine. Here we evaluate the feasibility of transdermal iontophoretic extraction of phenylalanine in vivo. Phenylalanine was iontophoretically extracted from the skin of healthy volunteers and of patients with phenylketonuria for up to 6 h and concentrations were compared with those measured in plasma. The amount of phenylalanine iontophoretically extracted from the skin declined over time, suggesting contribution of phenylalanine from the skin in the initial extraction. Phenylalanine iontophoretically extracted from skin correlated with plasma phenylalanine levels at plasma levels above 300 micromol/L. This correlation supports the feasibility of iontophoretic phenylalanine extraction for monitoring phenylketonuria.

Ethanol effects on the stratum corneum lipid phase behavior.

The stratum corneum is considered to be the diffusional barrier of mammalian skin for water and most solutes. The intercellular lipid multilayer domains of the stratum corneum are believed to be the diffusional pathway for most lipophilic solutes. Fluidization of the lipid multilayers in the presence of ethanol is frequently conceived to result in enhanced permeation. Current investigations address the effect of ethanol on the phase behavior in terms of stratum corneum lipid alkyl chain packing, mobility and conformational order as measured by Fourier transform infrared (FTIR) spectroscopy. Phospholipid multilamellar vesicles were also studied as model systems. There appeared to be no effect of ethanol on either the solid-solid phase transition or the gel phase interchain coupling of the stratum corneum lipids. However, there was a reduction in the mobility of the alkyl chains in the presence of ethanol. Possible mechanistic relationships between the current FTIR spectroscopic results with available literature data of ethanol induced lipophilic solute penetration enhancement through the skin are discussed.

The stratum corneum lipid thermotropic phase behavior.

The stratum corneum, the outermost layer of mammalian skin, is considered the least permeable skin layer to the diffusion of water and other solutes. It is generally accepted that the intercellular lipid multilayer domain is the diffusional pathway for most lipophilic solutes. Fluidization of the lipid multilayers is believed to result in the loss of barrier properties of the stratum corneum. Current investigations address the lipid thermotropic phase behavior in terms of lipid alkyl chain packing, mobility and conformational order as measured by Fourier transform infrared (FTIR) spectroscopy. A solid-solid phase transition is observed with increased alkyl chain mobility followed by a gel to liquid-crystalline phase transition near 65 degrees C. These results further elucidate the role of lipid fluidity that may contribute to the transport properties of the stratum corneum.

Fluorescence anisotropy studies on the interaction of the short chain n-alkanols with stratum corneum lipid liposomes (SCLL) and distearoylphosphatidylcholine (DSPC)/distearoylphosphatidic acid (DSPA) liposomes.

Previously, the action of the short chain n-alkanols (from C1 to C5) and isopropanol as possible enhancers on the transport of lipophilic and polar/ionic permeants across hairless mouse skin was investigated. In the present study, the steady-state fluorescence anisotropy was measured as a means of estimating the changes in fluidity caused by the n-alkanols at different depths in the stratum corneum lipid liposomes (SCLL). Some selected experiments with the distearoylphosphatidylcholine (DSPC)/distearoylphosphatidic acid (DSPA) liposomes were performed for relative comparisons. The effects of the n-alkanols on polarity sensitive parameters such as fluorescence lifetimes, fluorescence quantum yield ratios, and emission maxima were studied in the SCLL. The polarity of the bilayer decreased as the fluorescent probe was placed closer to the bilayer center and the n-alkanols did not alter this gradient. Assessment of the depth-dependent effects of the n-alkanols using SCLL showed that most of the significant changes in fluidity induced by the n-alkanols were observed at intermediate depths (C2-C9) and there was little or no increase in fluidity in the deep hydrophobic region close to the bilayer center. These results suggest that the short chain n-alkanols work as effective 'fluidizing' agents at the intermediate depths (C2-C9) in the bilayer.

Delivery of liposome membrane-associated sterols through silastic membranes.

The transport of sterols incorporated into the lecithin bilayer of small unilamellar liposomes through a model membrane was studied. A two-chamber diffusion cell containing liposomes with incorporated [4-14C)cholesterol or beta-[4-14C]sitosterol in the donor chamber and liposomes with unlabeled cholesterol in the receiver chamber was used. The permeability coefficients of the sterols through silastic rubber membranes which served as a model membrane were measured. The permeability for cholesterol incorporated into liposomes in a phosphatidyl choline/cholesterol molar ratio of 1 : 1, produced by sonication for 1 h, and subsequent centrifugation at 100 000 X g for 1 h, was 1.6 . 10(-8) cm sec-1. Dilution of the liposome suspension did not change the permeability coefficient significantly. The permeability coefficient of sitosterol incorporated into liposomes was about 4-times smaller than that of cholesterol. These results suggest that the sterols were delivered to the silastic membrane by the intact liposomes and that free solute was not involved in the transport to the membrane to a significant degree. The large differences in the permeability coefficients between cholesterol and sitosterol indicate that an aqueous interfacial barrier was crossed by the sterol during the delivery to the membrane.

A comparative study of the metastable equilibrium solubility behavior of high-crystallinity and low-crystallinity carbonated apatites using pH and solution strontium as independent variables.

UNLABELLED: Using solution strontium and pH as independent variables, the metastable equilibrium solubility (MES) behavior of two carbonated apatite (CAP) samples has been examined, a high-crystallinity CAP (properties expected to be similar to dental enamel) and a low-crystallinity CAP (properties expected to be similar to bone mineral). CAP samples were prepared by precipitation/digestion: (CAP A: high-crystallinity, 1.3 wt% CO3, synthesized at 85 degrees C; CAP B: low-crystallinity, 6.4 wt% CO3, synthesized at 50 degrees C). Baseline MES distributions were determined in a series of 0.1 M acetate buffers containing only calcium and phosphate (no strontium) over a broad range of solution conditions. To assess the influence of strontium, MES profiles were determined in a similar fashion with 20, 40, 60, and 80% of the solution calcium being replaced on an equal molar basis by solution strontium. To determine the correct function governing CAP dissolution, ion activity products (IAPs) were calculated from the compositions of buffer solutions based on the hydroxyapatite template (Ca(10-n)Sr(n)(PO4)6(OH)2 (n = 0-10)) and the calcium/hydroxide deficient hydroxyapatite template (Ca(9-n)Sr(n)(HPO4)(PO4)5OH (n = 0-9)). FINDINGS: (a) for CAP A, at high solution strontium/calcium ratios, the MES profiles were essentially superimposable when the solution IAPs were calculated using the stoichiometry of Ca6Sr4(PO4)6(OH)2 and for CAP B by a stoichiometry of Ca7Sr2(HPO4)(PO4)5OH; (b) for CAP A, at low strontium/calcium ratios, the stoichiometry yielding MES data superpositioning was found to be that of hydroxyapatite and for CAP B, that of calcium/hydroxide deficient hydroxyapatite. When other stoichiometries were assumed, good superpositioning of the data was not possible.

Calcium-level responsive controlled drug delivery from implant dosage forms to treat osteoporosis in an animal model.

The effects of plasma calcium levels on estradiol release from a self-setting apatite bone cement containing 0.5% estradiol and on the bone mineral density (BMD) of ovariectomized rats were investigated. Apatite cement consisting of an equimolar mixture of tetracalcium phosphate, dicalcium phosphate dihydrate and 0.5% beta-estradiol was prepared. The in vitro release profiles from the cements in simulated body fluid containing 0, 5 and 10 mg/100 ml calcium indicated that estradiol release rate decreased with increasing calcium concentration in the dissolution medium. After subcutaneous implantation of the cement, in vivo estradiol release in diseased rats (ovariectomized rats on a low calcium diet) was significantly higher than that in normal rats. The diseased rats maintained a low calcium level during drug release. The bone mass of the recovery model rat was greater after the experiment than before. The results suggested that the severity of osteoporosis in this animals can be reduced by the implantation of this estradiol-loaded apatite cement.

Physicochemical study of percutaneous absorption enhancement by dimethyl sulfoxide: dimethyl sulfoxide mediation of vidarabine (ara-A) permeation of hairless mouse skin.

Dimethyl sulfoxide's (DMSO) concentration-dependent influences on its own permeation rate through hairless mouse skin and on the concurrent permeation rates of water and the antiviral drug vidarabine (ara-A) have been studied at 37 degrees C using in vitro diffusion cells. Solubilities of ara-A in DMSO-water mixtures were also determined in order to assess ara-A's relative thermodynamic activity in the binary solvent media used in the mass transfer studies. Solubilities increased exponentially with increasing percentages of DMSO. Activity coefficients decreased accordingly. When the same DMSO medium was placed in each side of diffusion cell (balanced solvent configuration) permeability coefficients for ara-A decreased exactly as ara-A's solubility increased up to a 50% DMSO concentration, indicating the observed decreases in the mass transfer coefficients have thermodynamic origins. When DMSO media were placed in either the donor or receiver side of the cell up to the same 50% concentration point and opposed by a normal saline medium on the other side (asymmetric solvent configurations), the permeability of ara-A did not decrease and at some DMSO levels was substantially increased, behavior in marked departure from thermodynamic control. The behavior disparity between the 2 configurations of the cell suggests that cross-currents of solvents play a role in permeability enhancement. Regardless of solvent configuration, permeability coefficients for ara-A at 90 and 100% DMSO strengths were exaggeratedly large, consistent with severe impairment of the stratum corneum. Similar overall permeability behavior was observed for the 2 solvents, water and DMSO. Possible underlying mechanisms for these effects and the relative importance of the various mechanisms of DMSO enhancement as a function of DMSO's concentration and configuration are discussed.

Permeability of thermally damaged skin: I. Immediate influences of 60 degrees C scalding on hairless mouse skin.

Freshly sacrificed hairless mice were burned dorsally by direct contact with 60 degrees C water for periods ranging from 15 seconds to 8 min. Wounds ranging in degree from superficial epidermis damage to injury penetrating well into subcutaneous musculature were inflicted. Burned skin sections and reference abdominal skin sections were excised, placed in diffusion cells and investigated with regards to their permeabilities to water, methanol, ethanol, n-butanol and n-octanol. The data were couched in terms of ratios of permeability coefficients of burned skin to normal skin (scalding coefficients) for the same animal. Scalding increased permeability of skin to all compounds studied but the effects leveled out by 60 seconds. Protracted scalding was without great effect despite progressively increased depth of damage to the tissue as noted in histological sections. The degree of lost barrier competency attributable to 60 degrees C scalding was not marked for any compound but was definitely different for different alkanols. An approximately 3-fold permeability increase was noted with n-butanol, the most affected compound. The data demonstrate that near instantaneous alterations in permeability of skin accompany scalding, that decreased barrier competency does not correlate with the severity of a burn as measured in depth of the burn, and that thermal alteration of permeabilities is dependent on the physicochemical characteristics of the permeants.

Hydration and percutaneous absorption: I. Influence of hydration on alkanol permeation through hairless mouse skin.

A method to study the influence of hydration on skin permeability where the skin is immersed in saline for up to 30 hr and under circumstances where a steady state rate of permeation can be established in several minutes is indicated. These circumstances allow multiple, sequential runs over a period where the permeability coefficients of some chemicals are gradually changing. It has been found that the permeabilities of water, methanol and ethanol are little affected by such hydration. However, there is a doubling of the permeability coefficients of butanol and hexanol during the first 10 hr of immersion. More hydrophobic alkanols seem to be less sensitive to the protracted aqueous conditioning. In general the results indicate that there are complex molecular structure-permeability relationships operating in skin. More specifically, the hydration effects are insightful with respect to developing barrier models for skin as they are further indications that different parallel diffusional paths are followed by polar and semi- and nonpolar species.

An experimental skin sandwich flap on an independent vascular supply for the study of percutaneous absorption.

Further insights into the composite interactive processes of topically applied agents and percutaneous absorption and metabolism by functional skin in vivo have been hampered by the lack of a model system wherein the blood flow to and from the skin is independent but experimentally accessible. Utilizing microsurgical techniques, split-thickness skin grafting with syngeneic skin grafts, and the congenitally athymic (nude) rat, a skin sandwich flap system has been generated that has an independent but accessible vasculature and thus fills this void. We describe the methodology that has been developed to create the flap and present experiments that: demonstrate a lack of significant collateral circulation; quantify the microcirculation of the skin sandwich flap, host side, and graft side at various times during and after the flap has been generated, and note that blood flow to the flap is basically unchanged from host skin; demonstrate the utility of the system in measuring the amount of [14C]benzoic acid that appears in the flap when deposited on the surface in volatile and nonvolatile vehicles as a function of time; and demonstrate the fact that the flap can be reused, and that the total amount of [14C]benzoic acid absorbed across skin does not change in a substantial way as the flap ages.

A rice glutelin and a soybean glycinin have evolved from a common ancestral gene.

A cDNA clone covering the entire coding region for a glutelin subunit precursor has been identified from a library of endosperm-developing rice cDNA clones using a mixed oligodeoxynucleotide probe, and then by immunoprecipitation of hybrid-selected translation product with an antiserum against the acidic polypeptides of the glutelin. Analysis of the cDNA insert revealed that rice glutelin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs, like glycinin precursors of soybean. By comparing the predicted protein sequence of this precursor from monocots with that of glycinin A1aB1b precursor from dicots, it was found that the overall 32% of the amino acid positions are identical in both proteins. Because regions which show identities are dispersed throughout both molecules, the similarity is not due to convergent evolution, but to divergence evolution from a common ancestral gene.

Expression of soybean glycinin subunit precursor cDNAs in Escherichia coli.

As the cDNAs encoding A1aB1b and A2B1a subunit precursors of the glycinin A2 subfamily contain a unique NcoI site sequence, (A)CCATGG, occurring at their translation initiation sites, plasmids were constructed to direct the synthesis of those precursor proteins by inserting NcoI/PstI fragments derived from those cDNA clones into the NcoI/PstI-pKK233-2 expression vector in Escherichia coli MV1190, respectively. The resultant plasmids directed the expression of 57-kDa protein components that have molecular masses in agreement with those of the in vitro translation products directed by glycinin A2 subfamily mRNAs, by the addition of isopropyl beta-D-thiogalactoside. These proteins, which comprised as much as 1% of the total bacterial protein, are immunoprecipitable with rabbit antibodies specific for glycinin subunits. This procedure makes glycinin subunits available as a model for studying structure-function relationships in seed proteins using site-directed mutagenesis. This is the first expression of glycinin-like storage protein in E. coli.

Synthesis and evaluation of a series of 2'-O-acyl derivatives of 9-beta-D-arabinofuranosyladenine as antiherpes agents.

A series of four 9-(2-O-acyl-beta-D-arabinofuranosyl)adenines (5a-d) was synthesized by acylation of 9-[3,5-bis-O-(tert-butyldimethylsilyl)-beta-D-arabinofuranosyl]adenine (2), followed by removal of the tert-butyldimethylsilyl groups under conditions (HOAc, tetra-n-butylammonium fluoride) that prevented acyl migration. The four 2'-O-acyl derivatives 5a-d showed activity in vitro against herpes type 1 viruses [virus ratings = 1.5-2.6; MIC50 = 26-72 micrograms/mL (8.48-21.3 X 10(-5) M)]. The 2'-O-acetyl (5a) and 2'-O-valeryl (5d) derivatives were evaluated in a guinea pig model for genital herpes (herpes type 2); only 5a showed potent activity when given 6 or 24 h postinfection.

Factor VII Toyama (Thr 359 Met): a homozygous missense mutation causing severe type I deficiency.

We performed a DNA analysis on a patient with severe type I factor VII deficiency by the polymerase chain reaction amplification and a direct DNA sequencing method. The proband was a 66-year-old Japanese woman who had recurrent episodes of excessive bleeding after dental extraction. The functional and antigenic levels of plasma factor VII markedly reduced to 1.6% and 2% of normal, respectively. However, she had no serious symptoms such as intracranial or intraarticular hemorrhage. The analysis revealed that the patient was homozygous for a missense mutation, Thr (ACG) to Met (ATG) at codon 359 in the catalytic domain. Her deceased parents were first cousins, and their consanguineous marriage presumably resulted in the homozygosity in her. This patient was the first case of homozygote for the Thr359Met mutation, though heterozygotes for the mutation were previously found in an Italian family.

Mutations of the platelet thromboxane A2 (TXA2) receptor in patients characterized by the absence of TXA2-induced platelet aggregation despite normal TXA2 binding activity.

Previously, we reported five cases of platelet dysfunction characterized by the absence of thromboxane A2 (TXA2) - induced platelet aggregation despite normal TXA2 binding activity. In this platelet disorder, patients were divided into two groups; i.e. those whose platelets lacked or did not lack phospholipase C (PLC) activation (Group A and Group B, respectively) (Thromb Haemost 1996; 76: 1080). Furthermore, in one of the patients, we showed that a single amino acid substitution (Arg60 to Leu) in the first cytoplasmic loop of the TXA2 receptor (TXR) was responsible for this platelet disorder. However, mutational analysis of the TXR in the remaining patients has not been performed. Based on this background, we investigated the mutations of the TXR in these patients, and found that all of the patients have the same abnormality of the TXR (Arg60-->Leu), although the Group A patients were homozygous and the Group B patients were heterozygous for this mutation. This mutation is the only abnormality which has been found in this platelet disorder, and in patients heterozygous for this mutation, the mutant type TXR suppresses wild-type receptor-mediated platelet aggregation by a mechanism independent of PLC activation.