Biomedical subjects

K Funato

Publications and source records attributed to K Funato.

16 recordsLinked to original sources

PubMed2000

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.

Acyltransferases↗

PubMed2000

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.

Adult↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

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.

Adult↗

PubMed1997

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.

Adenosine Triphosphate↗

PubMed1997

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.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

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.

Animals↗

PubMed1996

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.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

Plasma factor triggering alternative complement pathway activation by liposomes.

Several plasma components, such as complement (C) components, play a role in the clearance of liposomes from the circulation. The interactions between liposomes and the C system were investigated in this study. Multilamellar vesicle (MLV) liposomes, which were damaged by activation of the complement, became susceptible depending on the density of cetylmannoside (Man) on the liposome membrane, and activation proceeded through the alternative C pathway as observed for liposomes without Man (PC-MLV) (K. Funato et al., Biochim. Biophys. Acta 1103:198-204, 1992). In addition, the capacity of Man-modified liposomes (Man-MLV) to activate the alternative C pathway was abolished by preadsorption of plasma with Man-MLV but not with PC-MLV. The results suggest that a specific plasma factor adsorbed with Man-MLV was responsible for the augmentation of the C activation and, further, that the rapid clearance of Man-MLV from the circulation is caused by both enhanced C-mediated liposome permeability and enhanced C-mediated phagocytosis of liposomes.

Animals↗

PubMed1994

Enhanced hepatic uptake of liposomes through complement activation depending on the size of liposomes.

The objective of this study was to differentiate the roles of opsonins and phagocytic cells in the size-dependent hepatic uptake of liposomes in the submicron region. The extent of opsonization decreased with the decrease in size of liposomes (from 800 to 200 nm in diameter) and no enhancement of uptake was observed at 200 nm. There was no effect of liposome size on the uptake of unopsonized liposomes. Serum was pretreated with empty liposomes of each size and its opsonic activity was measured in the perfused liver. The small liposomes could not consume the opsonic activity, while the larger ones did so substantially. These results suggest that opsonins bind to liposomes depending on the size of liposomes and phagocytic cells take up liposomes in proportion to the extent of opsonization. Size-dependent liposome degradation in serum was also found, which was consistent with the size-dependent complement activation, because liposomes with this composition have been shown to be degraded by complement. The mechanism of opsonization was examined by treating serum at 56 degrees C for 30 min or with anti-C3 antiserum. Since both treatments inhibited the opsonic activity, the hepatic uptake of liposomes is considered to occur via complement receptor. In conclusion, the size of liposomes affected complement recognition, and the liposomes were taken up by the liver depending on the extent of opsonization.

Animals↗

PubMed1994

The complement- but not mannose receptor-mediated phagocytosis is involved in the hepatic uptake of cetylmannoside-modified liposomes in situ.

In the elimination of injected liposomes in vivo, it is considered that several serum components play an important role on hepatic uptake of them. This study was conducted to clarify the hepatic uptake mechanism of cetylmannoside (Man)-modified multilamellar vesicles (Man-MLV) using perfused rat liver. In the presence of serum, Man-MLV was taken up by the liver depending on the serum concentration, and it showed an approximately two-fold higher accumulation than MLV without any surface modifications (PC-MLV). These hepatic uptakes of liposomes were obviously inhibited by preheating the serum at 56 degrees C for thirty minutes or by the treatment with anti-rat C3 antiserum. Further, SDS-PAGE followed by immunoblot analysis showed the deposition of iC3b on the opsonized Man-MLV. These results obtained in the present study suggested that hepatic uptake of Man-MLV was mainly mediated by complement receptor rather than mannose receptor on Kupffer cells in vivo.

Animals↗

PubMed1992

Contribution of complement system on destabilization of liposomes composed of hydrogenated egg phosphatidylcholine in rat fresh plasma.

Large multilamellar vesicles (MLV) composed of hydrogenated egg phosphatidylcholine (HEPC), cholesterol (CH), and dicetyl phosphate (DCP) rapidly release part of an entrapped aqueous marker when incubated with fresh rat plasma and thus have severely limited usefulness as drug carriers. The mechanisms causing the instability of liposomes in plasma were investigated in this study. The leakage of liposomal constituents was completely inhibited by pre-heating at 56 degrees C for 30 min with plasma or by treating with EDTA, K-76COOH, or anti-C3 antiserum but was not inhibited with EGTA/MgCl2. These results indicated that the destabilization of liposomes in fresh rat plasma was induced by activation of the alternative complement pathway (ACP). Furthermore, the complement third component (C3) was detected from the liposomes incubated with fresh plasma by SDS-PAGE followed by Western blotting and immune detection. The C3b deposited on the liposomal surface via ACP was rapidly cleaved to iC3b. The results obtained in the present study suggest a possibility that the liposomes composed of HEPC (without any surface modification) may be effective carriers for macrophages because C3b and its degradative products, iC3b are related to the opsonic function on phagocytosis of foreign particles by macrophages.

Animals↗

PubMed1992

The effects of resistance training on muscle area and strength in prepubescent age.

The purpose of this study was to determine the effect of strength training in prepubescent boys and girls on muscle strength and cross-sectional area of upper arm. Subjects were ninety-nine healthy elementary school children who belonged to 1st, 3rd and 5th grades and they were assigned to two groups of training (N = 52) and control (N = 47). Training group participated in strength training program for 12 weeks which consisted of maximum sustained isometric contraction of elbow flexion for ten sec, whereas control group did not participate in special training program in this period. In orger to determine the changes due to training, cross-sectional areas of the tissues in upper arm, such as muscle, fat and bone, were measured by the ultrasonic method. Maximum isometric and isokinetic muscle strength of elbow flexion and extension were measured by means of isokinetic cybex dynamometer. In order to assess the development of physiological maturity, TW2 method was used to estimate the skeletal age in each subject by taking the hand-wrist X-ray photograph. After 12-week training period, the whole cross-sectional areas increased in both training and control groups. This increment was due to significant increases in muscle and bone area in the training group and, on the other hand, due to the increase in fat area in the control. The increment of muscle area of training group was about 50% of that derived from the study on adults (Fukunaga, T., 1978). The increment in cross-sectional area of muscle with training was significantly correlated with the skeletal age.(ABSTRACT TRUNCATED AT 250 WORDS)

Arm↗

PubMed1989

Characteristics of leg extensor muscle in a world champion masters jumper. A case study.

To examine the characteristics of a world champion masters jumper (aged 71 yr), a study was undertaken which included a muscle biopsy and measurement of mechanical force production. Comparisons were made to biopsy data obtained from the literature and to force data on 7 active males (63-81 yrs). Cross-sectional areas of vastus lateralis muscle of the jumper's dominant thigh were 3.43 (type I), 3.32 (type IIA) and 2.34 (type IIB) microns 2 X 10(3). This is smaller than what is reported from sedentary young and old. The torque-time curve of maximal isometric knee extension indicates that the jumper had the highest value per body weight from the onset to the peak (3.48 Nm/kg; 58.2% higher than age-matched controls). These results suggest that the daily training of the jumper has a specific effect on the rate of force production despite his relatively small muscle fiber areas.

Aged↗

PubMed1988

Aerobic threshold, anaerobic threshold, and maximal oxygen uptake of Japanese speed-skaters.

The purpose of this study was to investigate the physiologic and metabolic parameters of speed-skaters with different training regimes and performance level and examine some physiologic prerequisites for speed-skating. The subjects were 25 male speed-skaters including members of the 1984 Japanese National Speed Skating Team whose ages ranged from 19 to 25 years. Aerobic threshold (AerT), anaerobic threshold (AnT), and VO2max were determined during a progressive bicycle ergometer exercise. The power was increased by 12.25 W every 3 min to exhaustion. AerT was determined using gas exchange variables; nonlinear increase in VE and VCO2, and peak VO2.VE-1. AnT was estimated from breakaway VE and the onset of decrease in FECO2.VO2max was measured during another incremental exercise on a bicycle ergometer. Mean AerT, AnT, and VO2max for skaters (n = 25) were 2.47 +/- 0.36.min-1 (61.1 +/- 7.2 %VO2max), 2.93 +/- 0.33.min-1 (73.4 +/- 5.9 %VO2max), and 4.06 +/- 0.42.min-1, respectively. All-arounders had higher AerT values but the same VO2max as sprinters. AnT of all-arounders was significantly higher than those of sprinters. A significant difference between the top ten elite skaters and the other skaters (n = 15) was found only in VO2max expressed as l.min-1. However, no significant correlation was noted between measured physiologic variables (AerT, AnT, and VO2max) and performances expressed as mean velocities at various events.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗