The pleiotrophin-ALK axis is required for tumorigenicity of glioblastoma stem cells.

Increasing evidence suggests that brain tumors arise from the transformation of neural stem/precursor/progenitor cells. Much current research on human brain tumors is focused on the stem-like properties of glioblastoma. Here we show that anaplastic lymphoma kinase (ALK) and its ligand pleiotrophin are required for the self-renewal and tumorigenicity of glioblastoma stem cells (GSCs). Furthermore, we demonstrate that pleiotrophin is transactivated directly by SOX2, a transcription factor essential for the maintenance of both neural stem cells and GSCs. We speculate that the pleiotrophin-ALK axis may be a promising target for the therapy of glioblastoma.

The critical role of cyclin D2 in cell cycle progression and tumorigenicity of glioblastoma stem cells.

Cancer stem cells are believed to be responsible for tumor initiation and development. Much current research on human brain tumors is focused on the stem-like properties of glioblastoma stem cells (GSCs). However, little is known about the molecular mechanisms of cell cycle regulation that discriminate between GSCs and differentiated glioblastoma cells. Here we show that cyclin D2 is the cyclin that is predominantly expressed in GSCs and suppression of its expression by RNA interference causes G1 arrest in vitro and growth retardation of GSCs xenografted into immunocompromised mice in vivo. We also demonstrate that the expression of cyclin D2 is suppressed upon serum-induced differentiation similar to what was observed for the cancer stem cell marker CD133. Taken together, our results demonstrate that cyclin D2 has a critical role in cell cycle progression and the tumorigenicity of GSCs.

Profiles of muscularity in junior Olympic weight lifters.

AIM: This study aimed to investigate the muscularity of strength-trained junior athletes. METHODS: Muscle thickness (Mt) values at 10 sites (anterior forearm, anterior upper arm, posterior upper arm, chest, abdomen, back, anterior thigh, posterior thigh, anterior lower leg, and posterior lower leg) were determined in junior Olympic weight lifters (OWL, n=7, 15.1+/-0.3 y, mean+/-SD) and non-athletes (CON, n=13, 15.1+/-0.3 y) using a brightness mode ultrasonography. Skeletal age assessed with the Tanner-Whitehouse II method (20 hand-wrist bones) was similar in OWL (16.4+/-0.7 y) and CON (16.3+/-0.6 y). RESULTS: At the 6 sites (anterior forearm, anterior upper arm, posterior upper arm, chest, back and anterior thigh), OWL showed significantly greater Mt values than CON even in terms of Mt relative to body mass(1/3) Mt x BM(-1/3). On the other hand, there were no significant differences between the 2 groups in the Mt ratios of the anterior to posterior site in the upper arm, thigh and lower leg and those of the back to either the chest or abdomen in the trunk. For OWL only, skeletal age was significantly correlated to Mt x BM(-1/3) at the abdomen (r=0.869, p<0.05) and anterior thigh (r=0.883, p<0.05). CONCLUSIONS: The findings here indicate that 1) as compared to adolescent non-athletes, junior Olympic weight lifters show a greater muscularity in the upper body and anterior thigh without predominant development in either of anterior and posterior sites within the same body segment, 2) for junior Olympic weight lifters, the muscularity of abdominal and knee extensor muscles is influenced by the biological maturation.

Combination of 22-oxa-1,25-dihydroxyvitamin D(3), a vitamin D(3) derivative, with vitamin K(2) (VK2) synergistically enhances cell differentiation but suppresses VK2-inducing apoptosis in HL-60 cells.

We originally reported that vitamin K(2) (VK2) effectively induces apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. In addition, VK2 was shown to induce differentiation of leukemia cells when the cells were resistant against VK2-inducing apoptosis. A novel synthetic vitamin D(3)derivative, 22-oxa-1,25-dihydroxyvitamin D(3) (OCT: oxacarcitriol) shows a more potent differentiation-inducing ability among myeloid leukemia cells in vitro with much lesser extent of the induction of hypercalcemia in vivo as compared to the effects of 1alpha,25(OH)(2)D(3). In the present study, we focused on the effects of a combination of OCT plus VK2 on leukemia cells. Treatment of HL-60 cells with OCT for 72 h induces monocytic differentiation. A combination of OCT plus VK2 dramatically enhances monocytic differentiation as assessed by morphologic features, positivity for non-specific esterase staining, and cell surface antigen expressions. This combined effect far exceeds the maximum differentiation induction ability at the optimal concentrations of either OCT or VK2 alone. In addition, pronounced accumulation of the cells in the G0/G1 phase is observed by combined treatment with OCT plus VK2 as compared with each vitamin alone. In contrast to cell differentiation, caspase-3 activation and apoptosis induction in response to VK2 are significantly suppressed in the presence of OCT in HL-60 cells. These data suggest that monocytic differentiation and apoptosis induction of HL-60 cells are inversely regulated. Furthermore, pronounced induction of differentiation by combined treatment with VK2 plus OCT was also observed in four out of six cases of primary cultured acute myeloid leukemia cells in vitro, suggesting that VK2 plus OCT might be a potent combination for the differentiation-based therapy for acute myeloid leukemias.

Vesicular and nonvesicular transport of ceramide from ER to the Golgi apparatus in yeast.

Transport and sorting of lipids must occur with specific mechanisms because the membranes of intracellular organelles differ in lipid composition even though most lipid biosynthesis begins in the ER. In yeast, ceramide is synthesized in the ER and transferred to the Golgi apparatus where inositolphosphorylceramide (IPC) is formed. These two facts imply that ceramide can be transported to the Golgi independent of vesicular traffic because IPC synthesis still continues when vesicular transport is blocked in sec mutants. Nonvesicular IPC synthesis in intact cells is not affected by ATP depletion. Using an in vitro assay that reconstitutes the nonvesicular pathway for transport of ceramide, we found that transport is temperature and cytosol dependent but energy independent. Preincubation of ER and Golgi fractions together at 4 degrees C, where ceramide transport does not occur, rendered the transport reaction membrane concentration independent, providing biochemical evidence that ER-Golgi membrane contacts stimulate ceramide transport. A cytosolic protease-sensitive factor is required after establishment of ER-Golgi contacts.

Myelodysplastic syndrome accompanied by Addison's disease and multiple autoimmune phenomena: steroid therapy resolved cytopenias and all immune disorders.

We report here a patient with myelodysplastic syndromes (MDS), which was complicated with several autoimmune disorders and asymptomatic immunologic abnormalities. An 82-year-old woman with refractory anemia (RA) rapidly developed thrombocytopenia with the appearance of symptoms such as purpura, fatigue, anorexia, and weight loss. Furthermore, clinical examinations revealed that she also had Addison's disease, rheumatoid arthritis, and autoimmune hematological diseases such as thrombocytopenia and hemolytic anemia. However, the cytopenia and all autoimmune disorders were remarkably improved after she received steroid therapy.

Apoptosis/differentiation-inducing effects of vitamin K2 on HL-60 cells: dichotomous nature of vitamin K2 in leukemia cells.

We originally reported that vitamin K2 (VK2) analogs, including menaquinone 4 (MK4) but not vitamin K1, effectively induce apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. It has also been reported by others that VK2 showed the differentiation-inducing activity in leukemia cell lines. To investigate the discrepancy between apoptosis- and differentiation-inductions of leukemia cells by VK2 treatment, we used bcl-2 gene transfected HL-60 cells (HL-60-bcl-2) which resulted in five-fold over-expression of BCL-2 protein, and then compared the effects of MK4 to the control HL-60-neo cells. Seventy-two hours of exposure to various concentrations of MK4 resulted in growth inhibition of these cells in a dose-dependent manner (0.1-50 microM), however, HL-60-bcl-2 was less sensitive against MK4. MK4 potently induced apoptosis of HL-60-neo cells along with the depolarization of mitochondrial membrane potential and caspase-3 activation. Notably, HL-60-bcl-2 was almost completely resistant to apoptosis induction in response to MK4, although cell growth inhibition was still observed. In spite of the abrogation of apoptosis induction, about 90% of HL-60-bcl-2 cells were arrested in the G0/G1 phase within 48 h of exposure to 10 microM of MK4 accompanied by up-modulation of p27KIP1 expression. Concomitantly, HL-60-bcl-2 cells underwent monocytic differentiation. These data suggest that VK2 also shows the differentiation inducing effects on leukemia cells which are resistant against VK2-inducing apoptosis. The dichotomous nature of VK2 against leukemia cells appears to have clinical benefits for the treatment of patients with leukemias and myelodysplastic syndromes.

High-performance liquid chromatography with chemiluminescence detection of penbutolol and its hydroxylated metabolite in rat plasma.

This paper describes a new method of high-performance liquid chromatography with chemiluminescence detection for the analysis of penbutolol (PB) and its main metabolite, 4-hydroxy penbutolol (4-OH PB) in rat plasma. 4-Dimethylaminosulfonyl-7-(N-chloroformylmethyl-N-methyl) amino-2,1,3-benzoxadiazole (DBD-COCl) was used as a fluorogenic labeling reagent. A mixture of hydrogen peroxide and bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) in acetonitrile was used as a post-column chemiluminogenic reagent. The derivatives of PB and 4-OH PB with DBD-COCI were separated by isocratic effluent with 0.01 M imidazole buffer (pH 7.0)-acetonitrile within 10 min. The detection limits of the proposed method for PB and 4-OH PB were 9.9 and 15 fmol on column, respectively. After intravenous administration of PB in rats, its plasma concentration profiles of PB and 4-OH PB were determined by the proposed method. PB was demonstrated to be rapidly metabolized to 4-OH PB at the same rate as cardiac output.

Ergometry for estimation of mechanical power output in sprinting in humans using a newly developed self-driven treadmill.

An evaluation of mechanical power during walking and running in humans was undertaken after developing a specially designed running ergometer (RE) in which the subjects gripped the handlebar in front of them keeping both arms straight and in a horizontal position. Ten subjects participated in comparisons of the mean horizontal pushing force (MFam) on the handlebar with the mean horizontal ground reaction force (MFfp) recorded by force platform under the RE during five different constant speeds of walking or running and sprint running with maximal effort. Mechanical power developed during sprint running on the RE was compared with a 50 m sprint. Mean linear velocity (Mv) of the RE belt was recorded by the rotary encoder attached to the axis of the belt. Mean mechanical power calculated from the handlebar setting (MPam = MFam x Mv) was compared to that calculated from force platform recordings (MPfp = MFfp x Mv). A high test-retest reproducibility was observed for both MFfp (r = 0.889) and MFam (r = 0.783). Larger values for the coefficient of variation for MFam (11.3%-15.8%) were observed than for MFfp (3.3%-8.2%). The MPam, which were obtained from five different constant speeds of walking, running and sprint running were closely correlated to those of MPfp (y = 0.98x - 19.10, r = 0.982, P < 0.001). In sprint running, MPam was 521.7 W (7.67 W.kg-1) and was correlated to the 50 m sprint time (r = -0.683, P < 0.01). It is concluded that the newly developed RE was useful in the estimation of mechanical power output during human locomotion such as when walking, jogging and sprinting.

Sphingoid base synthesis requirement for endocytosis in Saccharomyces cerevisiae.

The internalization step of endocytosis in yeast requires actin and sterols for maximum efficiency. In addition, many receptors and plasma membrane proteins must be phosphorylated and ubiquitylated prior to internalization. The Saccharomyces cerevisiae end8-1 mutant is allelic to lcb1, a mutant defective in the first step of sphingoid base synthesis. Upon arrest of sphingoid base synthesis a rapid block in endocytosis is seen. This block can be overcome by exogenous sphingoid base. Under conditions where endogenous sphingosine base synthesis was blocked and exogenous sphingoid bases could not be converted to phosphorylated sphingoid bases or to ceramide, sphingoid bases could still suppress the endocytic defect. Therefore, the required lipid is most likely a sphingoid base. Interestingly, sphingoid base synthesis is required for proper actin organization, but is not required for receptor phosphorylation. This is the first case of a physiological role for sphingoid base synthesis, other than as a precursor for ceramide or phosphorylated sphingoid base synthesis.

Measurement of specific movement power application: evaluation of weight lifters.

A new dynamometer was developed for the measurement of specific movement power (SMP) exerted by mono- or multi-articular movements. To determine the reproducibility of SMP, two identical test protocols were carried out on separate days for six movement types on seven adult males. The movement types were arm pull (AP), leg rise (LR), knee extension (KE), elbow flexion (EF), hip extension (HE) and squat movement (SQ). Variations in peak power obtained in two tests ranged from 0.7% (AP) to 9.6% (SQ). Coefficients of the test-re-test correlation in peak power ranged from 0.805 (SQ) to 0.961 (AP) and standard errors ranged from 4 W (EF) to 14 W (SQ). SMP in upper extremities increased from 166 W (EF) to 307 W (AP) resulting from the increase in velocity. However, in the movements of lower extremities, SMP increased from 506 W (KE) to 1351 W (SQ) as a result of the increase in force. To evaluate the validity of the SMP, a pull movement in weightlifting was tested and related to the athletic performance on weightlifters. Positive linear correlation (r = 0.862, p<0.001) was observed between SMP and the total weight best records. It is concluded that this newly developed dynamometer has enough reproducibility and validity for evaluating the SMP, which is developed by various joint movement patterns related to the sport. The feasibility of applying this measuring protocol to the testing and training programmes for improving the daily living activities and athletic performances should now be assessed.

Differences in muscle cross-sectional area and strength between elite senior and college Olympic weight lifters.

BACKGROUND: The purpose of this study was to investigate the profiles of muscle cross-sectional area (CSA) and strength capability in relation to lifting ability in Olympic weight lifters. METHODS: The subjects were 8 elite senior lifters (ESL, age=25.2+/-1.3 years, height=1.64+/-0.03 m, mass=68.6+/-4.2 kg, mean+/-SEM) and 9 college lifters (CL, 20.8+/-0.3 years, 1.67+/-0.03 m, 70.53.4 kg) whose predetermined weight classes were within the same range. The CSAs of elbow or knee extensor and elbow or knee flexor muscles were measured using a B-mode ultrasound apparatus. Concentric and eccentric maximal voluntary forces were determined with an isokinetic dynamometer at a constant velocity of 1.05 rad/sec. RESULTS: The best score of the total mass lifted in the snatch and the clean and jerk lifts was significantly higher in ESL than in CL even in terms of per unit of fat-free mass. There were no significant differences between the two groups in fat-free mass, muscle CSA and force values with the exception that ESL compared to CL showed significantly greater force in concentric knee flexion. However, the ratios of force to muscle CSA (F/CSAs) in concentric and eccentric elbow extensions, eccentric knee extension and concentric knee flexion were significantly higher in ESL than in CL. CONCLUSIONS: The present results indicated that the magnitude of muscular development in limbs was similar in elite senior and college lifters whose predetermined weight classes were within the same range. As compared to college lifters, however, elite senior lifters had a higher F/CSA in specific muscle action modes, which might relate to the optimal execution of the Olympic lifts.

A Salmonella virulence protein that inhibits cellular trafficking.

Salmonella enterica requires a type III secretion system, designated Spi/Ssa, to survive and proliferate within macrophages. The Spi/Ssa system is encoded within the SPI-2 pathogenicity island and appears to function intracellularly. Here, we establish that the SPI-2-encoded SpiC protein is exported by the Spi/Ssa type III secretion system into the host cell cytosol where it interferes with intracellular trafficking. In J774 macrophages, wild-type Salmonella inhibited fusion of Salmonella-containing phagosomes with lysosomes and endosomes, and interfered with trafficking of vesicles devoid of the microorganism. These inhibitory activities required living Salmonella and a functional spiC gene. Purified SpiC protein inhibited endosome-endosome fusion in vitro. A Sindbis virus expressing the SpiC protein interfered with normal trafficking of the transferrin receptor in vivo. A spiC mutant was attenuated for virulence, suggesting that the ability to interfere with intracellular trafficking is essential for Salmonella pathogenesis.

Resistance training affects GLUT-4 content in skeletal muscle of humans after 19 days of head-down bed rest.

This study assessed the effects of inactivity on GLUT-4 content of human skeletal muscle and evaluated resistance training as a countermeasure to inactivity-related changes in GLUT-4 content in skeletal muscle. Nine young men participated in the study. For 19 days, four control subjects remained in a -6 degrees head-down tilt at all times throughout bed rest, except for showering every other day. Five training group subjects also remained at bed rest, except during resistance training once in the morning. The resistance training consisted of 30 isometric maximal voluntary contractions for 3 s each; leg-press exercise was used to recruit the extensor muscles of the ankle, knee, and hip. Pauses (3 s) were allowed between bouts of maximal contraction. Muscle biopsy samples were obtained from the lateral aspect of vastus lateralis (VL) muscle before and after the bed rest. GLUT-4 content in VL muscle of the control group was significantly decreased after bed rest (473 +/- 48 vs. 398 +/- 66 counts. min-1. microgram membrane protein-1, before and after bed rest, respectively), whereas GLUT-4 significantly increased in the training group with bed rest (510 +/- 158 vs. 663 +/- 189 counts. min-1. microgram membrane protein-1, before and after bed rest, respectively). The present study demonstrated that GLUT-4 in VL muscle decreased by approximately 16% after 19 days of bed rest, and isometric resistance training during bed rest induced a 30% increase above the value of GLUT-4 before bed rest.

Reconstitution of phagosome-lysosome fusion in streptolysin O-permeabilized cells.

We have reconstituted fusion between phagosomes and lysosomes in streptolysin O-permeabilized J774-E macrophages. Fusion was assessed by measuring the delivery of avidin-conjugated horseradish peroxidase pre-internalized into lysosomes to phagosomes containing biotinylated beta-glucuronidase-conjugated paramagnetic beads (1-2 microm). Fusion was dependent on energy and exogenously supplied cytosol. Phagosome-lysosome fusion was greatly inhibited when microtubules were depolymerized by nocodazole treatment, suggesting that fusion occurs via microtubule-dependent transport. Furthermore, fusion was inhibited by GTPgammaS and Rab GDP dissociation inhibitor. These results suggest that rab proteins are involved in the regulation of fusion. Lastly, anti-NEM-sensitive factor (NSF) antibodies inhibited fusion, and addition of recombinant NSF wild type partially restored the fusogenic activity, indicating that NSF is required for fusion between phagosomes and lysosomes.

Muscle architecture and function in humans.

The present study focused on architectural factors which are considered to influence the linkage of muscle fiber and joint actions. By means of real-time ultrasonography we can observe clearly and noninvasively in vivo the movement of fascicle and aponeurosis in human muscle and measure directly the changes in pennation angle and length of fascicle during muscle contraction. During dorsi and plantar flexion without load the movement of tendinous tissue in human tibialis anterior muscle (TA) appeared to synchronize with the displacement of the ankle joint, indicating that the muscle tendon complexes are stiff relative to the applied force, which is fairly small in the case of foot shaking motion. On the other hand, when the ankle joint was fixed and the TA contracted 'statically' the ultrasonic echo from deep aponeurosis in the TA was observed to move proximally, indicating the elastic component (i.e. mainly tendinous tissue) was stretched significantly by the contraction force of muscle. In the case of the kneejoint, a length of fascicle in vastus lateralis decreased by 18% with the extension of the knee passively from a 100 degrees flexed position. When the knee extensors contracted 'statically' the fascicle length decreased at every joint angles and its magnitude was greater (30%) when the knee was closer to full extension than (5%) at the flexed positions. The present results clearly show that the architecture of actively contracting muscle fibers differ considerably than that which occurs when movement is passively induced. The use of cadaver data in the study of architecture and modeling of muscle functions would result in inaccurate, and in some cases even erroneous results.

Rab7 regulates transport from early to late endocytic compartments in Xenopus oocytes.

Rab7 has been shown to localize to late endosomes and to mediate transport from early to late endosome/lysosome in mammalian cells and in yeast. We developed a novel assay to quantify transport from early to late endosomes using the Xenopus oocyte. Oocytes were pulsed with avidin after which the oocytes were incubated to allow avidin transport to a late compartment. The oocytes were then allowed to internalize biotin-horseradish peroxidase (HRP). The oocytes were then injected with test proteins and incubated further to allow transport of biotin-HRP from early endosomes to late endosomal/lysosomal compartments. Transport was quantified by assessing the formation of HRP-biotin-avidin complexes. Injection of Rab7:wild-type (WT) and Rab7:Q67L, a GTPase defective mutant, stimulated transport. Rab5:WT had no effect. Rab7:WT-stimulated transport was inhibited by nocodazole, suggesting a role for intact microtubules. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocked Rab7:WT-stimulated transport, but Rab7:Q67L-stimulated transport was unaffected by the drug. Rab7:Q67L is constitutively activated and may not require phosphatidylinositol 3-kinase activity for activation. Rab7-stimulated transport requires N-ethylmaleimide-sensitive factor (NSF) activity as transport was blocked by N-ethylmaleimide and ATPase defective NSF mutants. Our results indicate that sequentially acting endocytic Rab GTPases utilize similar factors although their modes of action may be different.

Sequential actions of Rab5 and Rab7 regulate endocytosis in the Xenopus oocyte.

To explore the role of GTPases in endocytosis, we developed an assay using Xenopus oocytes injected with recombinant proteins to follow the uptake of the fluid phase marker HRP. HRP uptake was inhibited in cells injected with GTPgammaS or incubated with aluminum fluoride, suggesting a general role for GTPases in endocytosis. Injection of Rab5 into oocytes, as well as Rab5:Q79L, a mutant with decreased GTPase activity, increased HRP uptake. Injection of Rab5:S34N, the dominant-negative mutant, inhibited HRP uptake. Injection of N-ethylmaleimide-sensitive factor (NSF) stimulated HRP uptake, and ATPase-defective NSF mutants inhibited HRP uptake when coinjected with Rab5:Q79L, confirming a requirement for NSF in endocytosis. Surprisingly, injection of Rab7:WT stimulated both uptake and degradation/activation of HRP. The latter appears to be due to enhanced transport to a late endosomal/prelysosomal degradative compartment that is monensin sensitive. Enhancement of uptake by Rab7 appears to function via an Rab5-sensitive pathway in oocytes since the stimulatory effect of Rab7 was blocked by coinjection of Rab5:S34N. Stimulation of uptake by Rab5 was blocked by Rab5:S34N but not by Rab7:T22N. Our results suggest that Rab7, while functioning downstream of Rab5, may be rate limiting for endocytosis in oocytes.

Changes in force-velocity and power output of upper and lower extremity musculature in young subjects following 20 days bed rest.

Ten healthy sedentary students were exposed to 20 days bed rest (BR) to study the effect of simulated weightlessness on force(F)-velocity(V) characteristics and power(P) output of upper and lower limb movements. F, V and P were measured using a special dynamometer applicable to single joint movements [elbow flexion(EF) and extension(EE), knee flexion(KF) and extension(KE), and hip flexion] or multi-joint movements (squatting). Physiological cross-sectional areas(PCSA) of KF and KE muscles were measured by a magnetic resonance imaging technique. After BR, attenuation in P were observed in lower limb movements (decreased by 19.8-43.6% for squatting, KF and KE), in upper limb movements on the other hand, the decreases in P were not significant (approximately -5% for EF and EE). Decrease in P in lower limb were more pronounced in heavier loaded conditions which were characterized by decreases in both F and V. For KF and KE, decreases in maximal static F (-18.9 to approximately -26.8%) were more pronounced than the decreases observed in the PCSA (approximately -7%), resulting in decreases in specific tension (-12.3 to approximately -22.1%). Neural excitation potentials to generate maximal muscle tension or P might also be influenced by weightlessness.

Biopharmaceutical evaluation of the liposomes prepared by rehydration of freeze-dried empty liposomes (FDELs) with an aqueous solution of a drug.

We have evaluated a method for preparation of a dispersion of liposomes encapsulating a drug, namely rehydration of freeze-dried empty (not containing drug) liposomes with an aqueous drug solution (FDEL method). In the present study, we characterized and compared this method with the conventional method using a lipid composition of DPPC-DPPG-cholesterol in a molar ratio of 27:3:20. Two hydrophilic compounds, [3H]-inulin and [3H]-mannitol, were used as model drugs. Liposomal preparations by the FDEL method had an encapsulation efficiency of 2.9 and 6.7% for [3H]-inulin and [3H]-mannitol, respectively, when rehydrated and incubated at 70 degrees C. Since non-specific adsorption of these markers to liposomal membrane is negligible, this method produces liposomes which encapsulate a drug in the intravesicular space. One-tenth of the marker encapsulated in the liposomes prepared by the FDEL method (F-liposomes) was released very rapidly on incubation with rat plasma, followed by the slow release of the remaining fraction thereafter. No such rapid-release phase was observed for the liposomes prepared by the conventional method (C-liposomes). This suggests the existence of two types of encapsulation, loose encapsulation and tight encapsulation, in F-liposomes at least. Pharmacokinetic parameters of marker encapsulated tightly in F-liposomes were comparable to those in C-liposomes. It is likely that amphipathic drugs such as doxorubicin are incorporated into liposomes more easily than inulin and mannitol when formulated by the FDEL method. These results therefore suggest that the FDEL method is useful in the preparation of a liposomal formulation of a drug.