Application of glucosylceramide-based liposomes increased the ceramide content in a three-dimensional cultured skin epidermis.

Ceramide is an intercellular lipid of the stratum corneum and is one of the most important components of the epidermal permeability barrier. Glucosylceramide (GlcCer), a ceramide precursor, was applied to three-dimensional skin culture to regulate ceramide. GlcCer/dimyristoylphosphatidylcholine (DMPC) = 4/4 (molar ratio and GlcCer/DMPC/dimyristoylphosphatidylglycerol (DMPG) = 4/4/1(molar ratio) liposomes were prepared by the thin-layer method. The particle diameters of GlcCer/DMPC and GlcCer/DMPC/DMPG liposomes were 124.0 ± 0.6 and 119.3 ± 18.9 nm, and the zeta potentials were 1.3 ± 0.3 and -19.9 ± 0.3 mV, respectively. Stability of these GlcCer liposomes was measured by transmission light scattering. Transmission light scattering of neutrally charged GlcCer (GlcCer/DMPC) liposomes increased in a time dependent manner. In contrast, negatively charged GlcCer (GlcCer/DMPC/DMPG) liposomes were not changed. ß-Glucocerebrosidase activity was measured in a cultured human skin model. Results confirmed that the cultured human skin model has ß-glucocerebrosidase activity. GlcCer/DMPC/DMPG liposomes were applied to the three-dimensional cultured human skin model, and ceramide NS, NP, AS, and AP were extracted from it. The various extracted ceramides were separated by high-performance thin-layer chromatography and quantified by a densitometer. The amount of ceramide AS only in the cultured skin model was significantly higher with the application of GlcCer-based liposomes than that of the nonapplication group, and was also dose dependent. Thus, GlcCer-based liposomes are useful for enriching the ceramide AS levels in a three-dimensional cultured skin model.

Effect of hyaluronan tetrasaccharides on epidermal differentiation in normal human epidermal keratinocytes.

OBJECTIVE: Hyaluronan (HA) plays a role in keratinocyte proliferation and differentiation. In addition, HA has been shown to have different biological activities depending on its molecular weight. It has been reported that HA-mediated CD44 activation regulates keratinocyte differentiation. Therefore, the aim of this study was to investigate the influence of HA tetrasaccharides (HA4) on the regulation of keratinocyte differentiation, CD44 gene expression and CD44-phosphorylated protein in human keratinocytes, and compare HA4 with high molecular weight HA. METHODS: Normal human epidermal keratinocytes (NHEKs) were treated at doses of 1 µg mL(-1) HA or HA oligosaccharides (HA4). After treatment, cell viability was checked using an MTT (3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Each differentiation marker and CD44 mRNA expression was detected by real-time PCR. Each differentiation marker and CD44-phosphorylated protein was assessed by Western blotting. RESULTS: Hyaluronan and HA4 showed no cytotoxicity up to a dose of 1 µg mL(-1) . On day 3 after HA4 treatment, each differentiation marker mRNA and K10 protein level was higher than that of the control. On day 9, late differentiation marker mRNA and protein levels were increased with HA and HA4 treatment. In addition, HA4 treatment increased the expression of CD44 mRNA, CD44-phosphorylated protein and intracellular calcium concentrations. HA4 enhanced keratinocyte differentiation and increased CD44-phosphorylated protein levels. CONCLUSION: HA4 may induce epidermal differentiation through phosphorylation of CD44.

Increase in ceramide level after application of various sizes of sphingomyelin liposomes to a cultured human skin model.

Sphingomyelin-based liposomes (SPM-L) that were sized (or not) by extrusion through a filter with pores of 100, 200, or 400 nm were applied to a three-dimensional cultured human skin model in order to evaluate which size of SPM-L was most effective at increasing its ceramide level. The diameters of the SPM-L in PBS were 102.7, 181.0, 224.0, and 380.1 nm. The diameters of the liposomes in the culture medium were 117.5, 199.2, 242.1, and 749.8 nm. The diameter of the small liposomes (<200 nm in diameter) did not change much, at least for 7 days. SPM-L in saline or culture medium were applied to the basal layer side or stratum corneum side of the cultured skin model, and ceramide II, III, V, and VI were then extracted from it. The extracted ceramide molecules were separated by HPTLC, and the concentration of each type of ceramide was quantified using a densitometer. When the small SPM-L (110 or 190 nm in diameter) were applied to the basal layer side, the levels of ceramide III and V were increased. When they were applied to the stratum corneum side, the levels of ceramide II, III, V, and VI were significantly increased compared to those of the PBS group, especially after the application of the small SPM-L (110 nm in diameter). Thus, the application of small SPM-L was useful for increasing the ceramide II, III, V, and VI levels of a cultured human skin model.

A massively multi-scale approach to characterizing tissue architecture by synchrotron micro-CT applied to the human placenta.

Multi-scale structural assessment of biological soft tissue is challenging but essential to gain insight into structure-function relationships of tissue/organ. Using the human placenta as an example, this study brings together sophisticated sample preparation protocols, advanced imaging and robust, validated machine-learning segmentation techniques to provide the first massively multi-scale and multi-domain information that enables detailed morphological and functional analyses of both maternal and fetal placental domains. Finally, we quantify the scale-dependent error in morphological metrics of heterogeneous placental tissue, estimating the minimal tissue scale needed in extracting meaningful biological data. The developed protocol is beneficial for high-throughput investigation of structure-function relationships in both normal and diseased placentas, allowing us to optimize therapeutic approaches for pathological pregnancies. In addition, the methodology presented is applicable in the characterization of tissue architecture and physiological behaviours of other complex organs with similarity to the placenta, where an exchange barrier possesses circulating vascular and avascular fluid spaces.

Changes of ROS during a two-day ultra-marathon race.

To assess oxidative stress (OS) induced by endurance exercise, concentrations of serum reactive oxygen species (ROS) were determined in 70 Japanese male amateur runners completing a two-day ultra-marathon race. Serum ROS levels were analyzed at three time points: before the race (baseline), after the 1st day race (mid-race), and after the 2nd day race (goal) (post-race). The means (SE) of ROS were 151.4(3.7) (U. CARR.), 168.7(4.4), and 156.8(4.4), respectively. Significant positive trends were noted between age and serum ROS concentrations at the three race points (p<0.05 for all). After adjusting for age, BMI and average monthly running distance, the baseline serum ROS concentrations were positively associated with completion times of the first-day race, in particular (p<0.05), suggesting that the concentrations may predict physical performance. The ROS production increased at mid-race (p<0.05), but the levels returned to baseline levels at post-race, indicating that an antioxidant defense system may develop post-race to reduce OS.

Real-time analysis of liposomal trafficking in tumor-bearing mice by use of positron emission tomography.

Long-circulating liposomes are known to accumulate passively in tumor tissues of tumor-bearing animals. To evaluate the in vivo behavior of such liposomes, we investigated the real-time liposomal trafficking by a non-invasive method using position emission tomography (PET). Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, and palmityl-D-glucuronide (PGlcUA) in a molar ratio of 4:4:1 were prepared in the presence of 2-[18F]fluoro-2-deoxyglucose ([2-18F]FDG). [2-18F]FDG-labeled liposomes sized by extrusion through a filter with various-sized pores were administered to mice bearing Meth A sarcoma, and a PET scan was performed for 120 min. Small-sized, long-circulating liposomes (100 nm in diameter) constructed with PGlcUA tended to accumulate in the tumor tissues. On the contrary, control liposomes (100 nm in diameter) containing dipalmitoylphosphatidylglycerol instead of PGlcUA accumulated in the liver. Large-sized PGlcUA-containing liposomes (> 300 nm) also accumulated in the liver, as well as in the spleen. Time-activity curves indicated that the small long-circulating liposomes (< 200 nm) transiently accumulated in the liver right after the injection but that the accumulation there decreased time-dependently. These data suggest that, although the majority of small long-circulating liposomes remain in the bloodstream, some extravasate once into the interstitial spaces in the liver re-enter the bloodstream again, and finally accumulate in the tumor tissues. This PET technique might be useful for studying real-time liposomal trafficking and for tumor imaging.

Antitumor activity of vincristine encapsulated in glucuronide-modified long-circulating liposomes in mice bearing Meth A sarcoma.

Liposomes modified with the uronic acid derivative palmityl-D-glucuronide (PGlcUA) have a long circulation time and tend to accumulate in the tumors of tumor-bearing mice. Taking advantage of this character, we investigated the therapeutic effect of vincristine (VCR) encapsulated in liposomes containing PGlcUA (dipalmitoylphosphatidylcholine/cholesterol/PGlcUA = 4:4:1 as a molar ratio) on tumor-bearing mice. VCR was loaded into liposomes by a remote loading method, and then free or liposomal VCR was injected intravenously into BALB/c mice bearing Meth A sarcoma implanted subcutaneously 5 days before hand. Single-dose administration of VCR (3.0 mg/kg) in PGlcUA-liposomes significantly suppressed tumor growth, and prolonged the survival time (T/C = 1.37). Furthermore, two-dose administration of the liposomes cured one third of the animals. The therapeutic effect of PGlcUA-liposomes was greater than that of control liposomes containing dipalmitoylphosphatidylglycerol instead of PGlcUA. PGlcUA-liposomes might thus be a useful tool for delivering antitumor agents to tumor tissues.

Induction of coenzyme A-dependent transacylation activity in rat liver microsomes by administration of clofibrate.

The effect of administration of clofibrate on the activity of coenzyme A-dependent (CoA-dependent) transacylation of 1-acyl-glycerophosphocholine (1-acyl-GPC) was examined in rat liver microsomes. Administration of clofibrate to rats increased the activity of Co-A-dependent transacylation of 1-[14C]acyl-GPC and the activity reached a value (8.37 nmol/min per mg protein) twice that in control rats (3.95 nmol/min per mg protein) without any changes in apparent Km values for CoA (1.2 microM in control and 1.0 microM in clofibrate-treated) and 1-acyl-GPC (33.4 microM in control and 27.8 microM in clofibrate-treated). The rate of CoA-dependent transfer of [14C]arachidonic acid (20:4) from 1-acyl-2-[14C]20:4-glycerophosphoethanolamine (GPE) or 1-acyl-2-[14C]20:4-glycerophosphoinositol (GPI) to 1-acyl-GPC (synthesis of 1-acyl-2-[14C]20:4-GPC) was also increased by treatment with clofibrate (1.9-fold and 1.5-fold increases, respectively). These results suggest that a CoA-dependent transacylation system of 1-acyl-GPC was induced by treatment with clofibrate.

Effect of serum protein binding on real-time trafficking of liposomes with different charges analyzed by positron emission tomography.

Liposomes have been used as carriers of various materials and as tools for gene transfer: for the latter purpose, positively charged liposomes are usually used. To evaluate the stability in the presence of serum and the in vivo behavior of such liposomes as well as those aspects of neutral and negatively charged liposomes, we investigated liposomal agglutinability in the presence of serum, serum protein binding to these liposomes, and real-time liposomal trafficking by a non-invasive method using positron emission tomography (PET). Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol without or with charged lipid were prepared in the presence of mannitol, and the turbidity change in the presence of serum was determined. Turbidity increase was not observed for so-called long-circulating liposomes, i.e., liposomes modified with glucuronic acid or with poly(ethylene glycol), or for negatively charged liposomes containing dicetyl phosphate (DCP), phosphatidylglycerol, or phosphatidylserine. On the contrary, a significant turbidity increase was observed when positively charged liposomes modified with stearylamine, stearyltrimethylammonium chloride or 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl bromide (DMRIE), which is known as a component of liposomes for gene transfer, were used. These liposomes were found to have bound a high amount of serum proteins after separation of unbound serum proteins by use of a spin column. The liposomal trafficking in vivo was determined for three kinds of liposomes, i.e., liposomes with DMRIE, those with DCP, and those without charged lipids. These liposomes were prepared in the presence of 2-[18F]fluoro-2-deoxy-D-glucose ([2-18F]FDG), and the [2-18F]FDG-labeled liposomes were administered to mice to perform PET scans. Positively charged liposomes containing DMRIE showed high accumulation in the liver compared with neutral and negatively charged liposomes. Since DMRIE-liposomes tended to aggregate in the presence of serum, and to be associated with serum protein, these characteristics may lead to the high uptake of DMRIE-liposomes by the liver.

Liposomal Arg-Gly-Asp analogs effectively inhibit metastatic B16 melanoma colonization in murine lungs.

Analogs of a synthetic peptide having the L-arginine-L-glycine-L-aspartic acid (RGD) sequence have been found to decrease metastatic colonization. To enhance the metastasis-suppressing efficacy of these analogs, we sought to stabilize these analogs and to prolong their circulation time by incorporating them into a liposomal formulation. Various structures of RGD analogs grafted to hydrophobic groups were synthesized and then incorporated into liposomes. Liposomes composed of distearoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol and appropriate RGD analogs were injected intravenously along with B16BL6 murine melanoma cells into mice. Liposomal RGD (0.6 mumol of the analog equivalent to ca. 200 micrograms RGD peptides) inhibited lung colonization up to 76%. This dose is an order of magnitude lower than that for comparable inhibition reported for free RGD. Multi-dose administration of liposomal RGD (0.15 mumol of the analog) also inhibited the spontaneous lung metastasis of cells from a primary tumor site of B16BL6 cells subcutaneously implanted into the footpad of mice. Taken together, our data indicate that liposomal RGD may serve as a useful anti-metastatic agent.

Discrepancies in dietary intakes and plasma concentrations of fatty acids according to age among Japanese female dietitians.

OBJECTIVE: To clarify the influences of age on dietary intakes and plasma concentrations of fatty acids (FAs) in Japanese female dietitians. SUBJECTS AND METHODS: In autumn 1996, we estimated dietary FA intakes based on 7 day weighed diet records and analyzed plasma FA concentrations in 79 healthy Japanese female dietitians, and investigated their relationships with age, dividing into three age groups (young (32-42 y), middle-aged (43-50 y) and elderly (51-66 y)). RESULTS: Dietary intakes of total FA, saturated FAs, monounsaturated FAs, n-3 polyunsaturated FAs (PUFAs) and alpha-linolenic acid (18:3n-3) were significantly highest in the middle-aged group, and lowest in the elderly. Similar trends were observed for dietary intakes of n-6 PUFAs and linoleic acid (18:2n-6), but there were no differences with regard to eicosapentaenoic acid (EPA; 20:5n-3), docosahexaenoic acid (DHA; 22:6n-3) and n-3 highly unsaturated FAs (HUFAs=EPA+22:5n-3+DHA). On the other hand, plasma concentrations of all FAs except for arachidonic acid (20:4n-6) demonstrated positive correlations with age. Moreover, plasma concentrations of EPA in all age groups, DHA in the elderly and n-3 HUFAs in the middle-aged and the elderly were all positively correlated with dietary intakes. CONCLUSIONS: We should take into account the influence of age on dietary habit and lipid metabolism when interpreting associations between dietary FA intakes and plasma FA concentrations.

Relative validity of a semi-quantitative food frequency questionnaire versus 28 day weighed diet records in Japanese female dietitians.

OBJECTIVE: To assess the relative validity of a semi-quantitative food frequency questionnaire (SQFFQ) against 28 day weighed diet records (WDRs). SUBJECTS AND METHODS: The SQFFQ was administered to 106 (21 male and 85 female) Japanese dietitians in Aichi Prefecture in autumn, 1996 and four-season consecutive 7 day WDRs were carried out during 1996-1997. We evaluated validity of intakes of 15 foods and 31 macro- and micro-nutrients based on the SQFFQ against those according to 28 day WDRs among 79 Japanese female dietitians. RESULTS: Mean daily intakes of selected foods and nutrients determined by the SQFFQ were generally equivalent to those measured by 28 day WDRs. Pearson's de-attenuated correlation coefficients (CCs) with log-transformation and energy-adjustment between intakes of selected foods and nutrients quantified by the SQFFQ and 28 day WDRs (minimum-median-maximum) ranged from 0.17 (beverages)-0.52 to 0.74 (rice), and Spearman's rank CCs with energy-adjustment ranged from 0.28 (confectionery)-0.42 to 0.68 (rice). Respective Pearson's CCs for intakes of nutrients were 0.28 (PUFAs)-0.51 to 0.73 (magnesium), and Spearman's rank CCs ranged from 0.23 (n-3 PUFAs)-0.45 to 0.71 (magnesium). Favorably higher agreement for intakes of foods/nutrients was achieved along with lower disagreement. CONCLUSIONS: Satisfactorily higher relative validity was attained in Japanese female dietitians with the SQFFQ. This calibrated questionnaire seems therefore appropriate for administration to Japanese dietitians to clarify associations between diet and health/disease. SPONSORSHIP: A grant-in-aid from the Ministry of Education, Science, Sports and Culture (06454242).

Foods contributing to absolute intake and variance in intake of selected vitamins, minerals and dietary fiber in middle-aged Japanese.

Using 351 one-day weighted diet records, we selected foods providing vitamins, minerals and dietary fiber according to contribution analysis (CA) and multiple regression analysis (MRA). Vitamin C was supplied by various vegetables and fruits, and carotene was specifically derived from green-yellow vegetables based on MRA as well as CA. Vitamin A was provided by green-yellow vegetables, fruits, chicken egg and milk (whole) according to CA; whereas chicken liver and pork liver were major sources according to MRA. Vitamin E was mainly of vegetable origin as determined by CA, and largely of spinach, safflower oil and pumpkin as determined by MRA. Vitamin D was mainly derived from chicken egg, fish and mushroom based on CA, and particularly from fish based on MRA. Calcium was supplied by milk (whole), soy products and chicken egg as determined by CA; while milk, tofu and various small fishes were the main contributors to variance. Magnesium was provided by soy products, well-milled rice and spinach according to both analyses, and iron by chicken egg, spinach and soy products. Zinc was largely derived from well-milled rice, followed by chicken egg and milk (whole) as determined by CA, and copper was provided by well-milled rice, soy and wheat products. Dietary fiber was supplied by vegetable sources, whether water soluble or insoluble, based on both analytic methods.

Scanning electron microscopy of fowl oviducal cells and HeLa and BHK-21 cells incubated with or without fowl spermatozoa in vitro.

The morphology of cells cultured at 41 C with and without fowl spermatozoa was observed with the scanning electron microscope. At the beginning of incubation, abnormal spermatozoa were comparatively few, and spermatozoa were found on the surface of the cultured cells. Morphological alteration of spermatozoa increased with increasing incubation interval and included head and tail coiling with occasional separation of heads from flagella.

[The effect of long-term group education for obese women in a public health center].

In order to improve maintenance of weight reduction, a long-term (5 month) weight reduction class for obese women consisting of repeated group learning three times a month in areas of dietary regimen and practice of physical exercise, and self-help group activities was conducted at a health center. Maintenance of achieved weight reduction was studied six months and one year after completion of the class. The results are as follows. 1) Among 30 alumni, percentages of rebound, regaining weight of more than half of reduction during the class, were 18% at six months and 48% at one year after the class. These results appear to indicate the difficulty in maintaining weight loss. 2) However, when changes were studied in terms of fat mass and lean body mass, rebound of fat mass, regaining fat mass of more than half of reduction, was very small--only 5%, through the follow-up periods--while lean body mass increased gradually. These results indicate very good maintenance of fat mass reduction, and from the view point of the objective of weight reduction which is principally fat mass reduction, this objective was supported. 3) The attendees were divided into two groups, diet group and exercise group. There were almost no differences in the follow-up results for weight maintenance between the two groups. This can be attributed to the fact that since the combination of dieting and exercise was emphasized in both groups, with moderate energy restriction and balanced food intake in the dietary regimen and aerobic walking for exercise in addition to the classroom learning, instructions for lifestyle changes were rather similar for both groups.

Changes of mental stress biomarkers in ultramarathon.

We investigated the possible influence of an exhaustive physical exercise on mental stress biomarkers (serotonin, tryptophan, and beta-endorphin) along with dopamine, noradrenaline and free fatty acids in an ultramarathon race in which 45 km was run on the first day and 90 km on the second. We obtained serum samples at 6 different time points during and after the race from 18 Japanese male runners who completed the marathon. Overall changes of serum serotonin and tryptophan concentrations were statistically significant according to ANOVA for repeated measurements (p < 0.05). Serum serotonin levels elevated rapidly on the first day with the post hoc Tukey's test. Tryptophan concentrations inversely decreased during the race, possibly because of utilization for synthesis of serotonin. Levels of beta-endorphin appeared to increase on the first and second days, but were not statistically significant. In conclusion, serum serotonin, tryptophan and beta-endorphin appeared to be used for mental stress markers in physical exercise.