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Introduction To investigate the biodistribution and retention properties of the new super paramagnetic iron oxide (new SPIO: mean hydrodynamic diameter, 100 nm) nanoparticles, which have concentrated polymer brushes in the outer shell and are difficult for phagocytes to absorb, and to compare the new SPIO with clinically approved SPIO (Resovist: mean hydrodynamic diameter, 57 nm). Materials and methods 16 male C57BL/6N mice were divided in two groups according to the administered SPIO (n = 8 for each group; intravenous injection does, 0.1 ml). In vivo magnetic resonance imaging (MRI) was performed before and one hour, one day, one week and four weeks after SPIO administration by two dimensional-the fast low angle shot (2D-FLASH) sequence at 11.7T. Ex vivo high-resolution images of fixed organs were also obtained by (2D-FLASH). After the ex vivo MRI, organs were sectioned and evaluated histologically to confirm the biodistribution of each particle precisely. Results The new SPIO was taken up in small amounts by liver Kupffer cells and showed a unique in vivo MRI contrast pattern in the kidneys, where the signal intensity decreased substantially in the boundaries between cortex and outer medulla and between outer and inner medulla. We found many round dark spots in the cortex by ex vivo MRI in both groups. Resovist could be detected almost in the cortex. The shapes of the dark spots were similar to those observed in the new SPIO group. Transmission electron microscopy revealed that Resovist and the new SPIO accumulated in different cells of glomeruli, that is, endothelial and mesangial cells, respectively. Conclusion The new SPIO was taken up in small amounts by liver tissue and showed a unique MRI contrast pattern in the kidney. The SPIO were found in the mesangial cells of renal corpuscles. Our results indicate that the new SPIO may be potentially be used as a new contrast agent for evaluation of kidney function as well as immunune function.

Fluorescent probes that emit in the near-infrared (NIR, 700-1,300 nm) region are suitable as optical contrast agents for in vivo fluorescence imaging because of low scattering and absorption of the NIR light in tissues. Recently, NIR quantum dots (QDs) have become a new class of fluorescent materials that can be used for in vivo imaging. Compared with traditional organic fluorescent dyes, QDs have several unique advantages such as size- and composition-tunable emission, high brightness, narrow emission bands, large Stokes shifts, and high resistance to photobleaching. In this paper, we report a facile method for the preparation of highly fluorescent, water-soluble glutathione (GSH)-coated NIR QDs for in vivo imaging. GSH-coated NIR QDs (GSH-QDs) were prepared by surface modification of hydrophobic CdSeTe/CdS (core/shell) QDs. The hydrophobic surface of the CdSeTe/CdS QDs was exchanged with GSH in tetrahydrofuran-water. The resulting GSH-QDs were monodisperse particles and stable in PBS (phosphate buffered saline, pH = 7.4). The GSH-QDs (800 nm emission) were highly fluorescent in aqueous solutions (quantum yield = 22% in PBS buffer), and their hydrodynamic diameter was less than 10 nm, which is comparable to the size of proteins. The cellular uptake and viability for the GSH-QDs were examined using HeLa and HEK 293 cells. When the cells were incubated with aqueous solutions of the GSH-QDs (10 nM), the QDs were taken into the cells and distributed in the perinuclear region of both cells. After 12 hrs incubation of 4 nM of GSH-QDs, the viabilities of HeLa and HEK 293 cells were ca. 80 and 50%, respectively. As a biomedical utility of the GSH-QDs, in vivo NIR-fluorescence imaging of a lymph node in a mouse is presented.

Arteriovenous (AV) fistulas have been used clinically for improving adjunctive bypass patency. Such AV shunting induces retrograde flow in the microvascular network, which may induce microvascular remodeling and angiogenesis at the chronic phase. This paper was aimed to examine heterogeneity of blood flow among capillaries in the retrograde microcirculation induced by AV shunting. An AV anastomosis was created in rat hind limb. Using a dual window method or frame-by-frame technique on the fluorescence microscopic video images, we measured velocities of red blood cells (RBCs) flowing in the capillary network in three flow conditions: control (normal flow), arterial occlusion, and AV shunting (retrograde flow). For each flow condition, RBC velocities were obtained in 155 capillaries of 6 rats. By classifying all the capillaries into four groups based on the levels of RBC velocity in the occlusion state, we evaluated the mean velocities, coefficient of variation (CV), and histograms for each group of capillaries. The mean velocity and CV in each group changed significantly from the control to AV shunting states. Especially, most significant changes appeared in capillary groups where the superficial femoral artery or its collateral arteries might have a direct influence. Though the AV shunting improved capillary perfusion in the mean level, major parts of capillaries still remained at low perfusion.

The dynamics behaviour and underlying principles of macrophages movement within living bodies still being unknown, we are currently studying unique sets of pictures in order to better understand them and develop a simulator able to successfully reproduce them. In previous works, we presented our early efforts in building a simple 2D macrophages simulator, as well as a new set of 3D data and a new tool to visualize them. In this paper, our goal is to progress towards a full analysis of our 3D data by extending our visualizing tool into an analysing tool, able to fully break the data about macrophages movement. By applying object recognition and object tracking techniques, we developed a tool able to display and record all information relative to the moves of a given three-dimensional macrophages set, information we will be able to use in order to develop a new 3D macrophages simulator.

Recent studies have demonstrated that immune cells play an important role in the pathogenesis of many neurological conditions. Immune cells constantly survey the brain microvasculature for irregularities in levels of factors that signal homeostasis. Immune responses are initiated when necessary, resulting in mobilisation of the microglial cells resident in the central nervous system (CNS) and/or of infiltrating peripheral cells. However, little is known about the kinetics of immune cells in healthy and diseased CNS, because it is difficult to perform long-term visualisation of cell motility in live tissue with minimal invasion. Here, we describe highly sensitive in vivo MRI techniques for sequential monitoring of cell migration in the CNS at the single-cell level. We show that MRI combined with intravenous administration of super-paramagnetic particles of iron oxide (SPIO) can be used to monitor the transmigration of peripheral phagocytes into healthy or LPS-treated mouse brains. We also demonstrate dynamic cell migration in live animal brains with time-lapse MRI videos. Time-lapse MRI was used to visualise and track cells with low motility in a control mouse brain. High-sensitivity MRI cell tracking using SPIO offers new insights into immune cell kinetics in the brain and the mechanisms of CNS homeostasis.

This paper demonstrates the possibility of in vivo imaging for neuroimmunological assessments using ultra high-field magnetic resonance imaging (UHF-MRI). UHF-MRI provides a highly sensitive MR microimaging technique could be used to identify previously invisible pathologies and cellular dynamics in the central nervous system (CNS) of living animals. Our technique could reveal the mechanisms underlying the immune responses and cell dynamics during neuroinflammation, CNS diseases, and also in the normal state.

Macrophages are well known cells in biology. While macrophage plays an important role in the immune system, the behavior rules of macrophages are unknown. In our research, the final goal is to find action rules for behavior of macrophages. Our current aim is to develop a simulator which can reproduce the movement of macrophages. As the first step, we try to develop a simulator that can imitate macrophages' behavior in three-dimensional space. In order to develop a simulator, some movies of actual macrophages in live mice are used. After parsing the movies, we try to develop a simulator which can imitate behaviors as same results as the observed. Our proposed simulator is based on cellular automata which are evolved by genetic algorithms. From the results of our simulator, we can see that the artificial macrophages behave as the similar movement of actual macrophages.

Animal models were developed for chronic observation with using two-photon microscopy. To demonstrate potency of the models, T-cell and macrophage migration in inguinal lymph node, spleen, abdominal cavity and kidney were observed for two weeks on single mice. The image quality was maintained high enough during an observation period to count and trace cell population and migration.

SUMMARYThe gas transport system of a bumblebee was investigated by measuring the oxygen partial pressure PO2 in the wing muscle. In the resting bee, PO2 showed a regular pattern of fluctuation with a typical period of 70–120s. Fluctuations in muscular PO2 were associated with intermittent abdominal pumping. Ventilation by abdominal movements may not be necessary during rest because PO2 is high (8.5–9.2kPa) in the anaesthetised bee. Thermal effects on muscular PO2 were examined by cooling the bee, causing the amplitude of PO2 fluctuations to increase. In most flight experiments, the bee started to fly after elevating muscle PO2 by abdominal pumping; muscle PO2 then decreased at the onset of flight. However, when a flight began without pre-flight ventilation, PO2 increased monotonically. During flight, muscle PO2 reached a mean level (6.36±1.83kPa) that was much higher than the lowest value recorded during discontinuous ventilation during rest. The bumblebee effectively uses abdominal movements to assist in convective gas transport not only during flight but also at rest.

Nitric oxide (NO) is a gaseous mediator involved in various physiological phenomena, such as vasorelaxation and neurotransmission. Investigation of local cellular responses of NO production in vivo and in vitro requires a measurement method with a high spatial resolution. For selective NO measurement, we therefore developed a microcoaxial electrode whose tip diameter is less than 10 microm. Calibration using various concentrations of NO (0.1-1.0 microM) showed that the electrode has good linearity (r = 0.99) and its detection limit is 0.075 microM (S/N = 3). We verified the applicability of this electrode to in vivo and in vitro local measurement NO released from bovine aortic cultured endothelial cells (BAECs) stimulated by acetylcholine (ACh). After the addition of ACh, a transient increase in NO concentration was detected by the electrode. In the presence of NG-nitro-L-arginine methyl ester (L-NAME), a putative NO synthase inhibitor, NO release (peak NO concentration) from RAECs was significantly less than that in the absence of L-NAME (0.18 +/- 0.04 microM vs 0.47 +/- 0.13; P < 0.01). After removal of L-NAME, NO release partially recovered (0.39 +/- 0.10 microM). In conclusion, the microcoaxial electrode was successfully applied to direct and continuous NO measurement in biological systems.

The morphological structure of vascular smooth muscle cells was analyzed in rabbit mesentery, using fluorescent images obtained by confocal laser scanning microscopy. The mesentery sections were stained with specific antibodies against myosin. The 3D image of terminal arterioles was reconstructed based on the sliced images. The resultant image shows, the arterioles were ovally collapsed in the transverse section. The arteriolar wall was consisted of spindle shaped endothelial cells aligned along the flow direction; surrounded by a mono-layer of spirally arranged smooth muscle cells. Individual smooth muscle cell surrounded the vessel to the extent of one to one and half loops. In between the smooth muscle spirals, several gaps were observed; while the endothelial cell arranged like a cobblestone. The dimension of the gap was equivalent to one or two smooth muscle cell width.

Insects augment oxygen supply using convective transport during flight in two ways: with deforming tracheae by surrounding muscles movement (muscle pumping) and with contracting air sacs by exoskeleton movement (abdominal or thoracic pumping). However, because induced flow inside tracheae is difficult to measure, it is not known how the convective transport actually contributes. By comparison between direct measurement of oxygen partial pressure in a flight muscle based on electrochemical method and flight/ventilation activities in a bumblebee, Bumbus hypocrita hypocruta, a method was developed for estimating gas transport efficiency. Oxygen partial pressure, PO2, in the bee periodically fluctuated with discontinuous abdominal movement in normal air. While the PO2 strongly varied among individuals in normal air, the PO2 took a unique value in oxygen poor air (l8%). By enhancing ventilation, the bee could respire in an oxygen poor atmosphere up to 2%. Furthermore, the bee could fly in an atmosphere of 6%, in which the PO2 decreased to 0.7%. Estimated efficiency of the gas transport increases with atmospheric oxygen concentration decreases.

A foraging bee is one of the most actively working insects. The bee's abdomen is well equipped with muscles for both contraction and extension. Besides, the bee has well developed air sacs not only in the abdomen but in the thorax. From view of the respiratory organ, a worker bee is a good example of a high oxygen consumed living thing. Here the gas transport system was investigated by measuring the oxygen partial pressure, Po2, in the bumble bee thorax. The measurement was made by directly inserting a needle electrode into a wing muscle. Results show the Po2 in a resting bee fluctuates with typically 70-120 sec interval. At phase of Po2 increase, the bee moved an abdomen repeatedly to ventilate air inside the body. The discontinuous ventilation should be beneficial for reducing water/heat loss but not necessary for oxygen supply at resting condition, therefore thermal effect to the ventilation pattern was examined, by which coiling a body changes the respiration from shallow to deep. In most case of the flight experiment, the bee started flying after elevating Po2 in the muscle by the abdominal movement, in that Po2 in the muscle decreased at onset of the flight. However, Po2 during flight gradually approaches the certain Po2 level which is much higher than the lowest value in the discontinuous ventilation. When the flight started without the preflight movement and Po2 was low in the muscle, Po2 inversely increased by the flight. To increase the oxygen supply during flight, the bee continuously contracted and extended the abdomen. The bee effectively uses the well equipped abdomen to take an advantage of the convective transport.

Fully developed intermittent flow in a strongly curved tube was numerically simulated using a numerical scheme based on the simpler method. Physiological pulsatile flow in the aorta was simulated as intermittent flow, with a waveform consisting of a pulse-like systolic flow period followed by a stationary diastolic period. Numerical simulations were carried out for the following conditions: Dean number κ=393, frequency parameter α=4–27, curvature ratio δ=1/2, 1/3 and 1/7, and intermittency parameter η=0–1/2, where η is the ratio of a systolic time to the cycle period. For α=18 and 27 the axial-flow profile in a systolic period becomes close to that of a sinusoidally oscillatory flow. At the end of the systole, a region of reversed axial velocity appears in the vicinity of the tube wall, which is caused by the blocking of the flow, similar to blocked flow in a straight tube. This area is enlarged near the inner wall of the bend by the curvature effect. Circumferential flow accelerated in a systole streams into the inner corner and collides at the symmetry line, which creates a jet-like secondary flow towards the outer wall. The region of reversed axial velocity is extended to the tube centre by the secondary flow. The development of the flow continues during the diastolic period for α higher than 8, and the flow does not completely dissipate, so that a residual secondary vortex persists until the next systole. Accordingly, the development of secondary flow in the following systolic phase is strongly affected by the residual vortex at the end of the previous diastolic phase, especially by stationary diastolic periods. Therefore, intermittent flow in a curved tube is strongly affected by the stationary diastolic period. For η=0 and 1/5, the induced secondary flow in a systole forms additional vortices near the inner wall, whereas for η=1/3 and 1/2 additional vortices do not appear. The characteristics of intermittent flow in a curved tube are also strongly affected by the length of the diastolic period, which represents a period of zero flow.

Magnetic resonance imaging (MRI) is widely employed for the diagnosis of multiple sclerosis (MS). However, sometimes, the lesions found by MRI do not correlate with the neurological impairments observed in MS patients. We recently showed autoreactive T cells accumulate in the fifth lumbar cord (L5) to pass the blood–brain barrier and cause inflammation in the central nervous system of experimental autoimmune encephalomyelitis (EAE) mice, an MS model. We here investigated this early event using ultrahigh-field MRI. T2-weighted image signals, which conform to the water content, increased in L4 and L5 during the development of EAE. At the same time, the sizes of L4 and L5 changed. Moreover, angiographic images of MRI showed branch positions of the blood vessels in the lower lumbar cords were significantly altered. Interestingly, EAE mice showed occluded and thickened vessels, particularly during the peak phase, followed by reperfusion in the remission phase. Additionally, demyelination regions of some MS patients had increased lactic acid content, suggesting the presence of ischemic events. These results suggest that inflammation-mediated alterations in the lower lumbar cord change the homeostasis of the spinal cord and demonstrate that ultrahigh-field MRI enables the detection of previously invisible pathological alterations in EAE.

Macrophages are an essential part of our immune system and one of the well-known cells in biology. However, despite its importance its usual behaviour and moving rules are mostly unknown due to the lack of observatory data for the macrophages. An experiment was conducted in an attempt to obtain new data by observing the macrophages in a living rat and produced a couple of movies. Our objective is to exploit these results to develop a simulator that is able to reproduce the moves of a macrophage population. After the movie analysis, we identified a few characteristics to implement into a first simple simulator. Although its performance is very limited, it represents a first step for further research of understanding macrophages.

Lymphocyte extravasation from the high endothelial venules (HEVs) of lymph nodes is crucial for the maintenance of immune homeostasis, but its molecular mechanism remains largely unknown. In this article, we report that lymphocyte transmigration across the basal lamina of the HEVs is regulated, at least in part, by autotaxin (ATX) and its end-product, lysophosphatidic acid (LPA). ATX is an HEV-associated ectoenzyme that produces LPA from lysophosphatidylcholine (LPC), which is abundant in the systemic circulation. In agreement with selective expression of ATX in HEVs, LPA was constitutively and specifically detected on HEVs. In vivo, inhibition of ATX impaired the lymphocyte extravasation from HEVs, inducing lymphocyte accumulation within the endothelial cells (ECs) and sub-EC compartment; this impairment was abrogated by LPA. In vitro, both LPA and LPC induced a marked increase in the motility of HEV ECs; LPC’s effect was abrogated by ATX inhibition, whereas LPA’s effect was abrogated by ATX/LPA receptor inhibition. In an in vitro transmigration assay, ATX inhibition impaired the release of lymphocytes that had migrated underneath HEV ECs, and these defects were abrogated by LPA. This effect of LPA was dependent on myosin II activity in the HEV ECs. Collectively, these results strongly suggest that HEV-associated ATX generates LPA locally; LPA, in turn, acts on HEV ECs to increase their motility, promoting dynamic lymphocyte–HEV interactions and subsequent lymphocyte transmigration across the basal lamina of HEVs at steady state.

Human aortic arch flow was regarded as an intermittent flow in a curved tube with large curvature ratio δ=1/3. The waveform of aortic flow was obtained by means of Doppler echocardiography from both resting and post-exercise conditions of healthy human adults (age : 22∼27). The result in the resting condition (Re=2300, α=13.34) shows that the transverse flow field is characterized by the appearance of three vertices (residue vortex from the previous cycle, one generated near the Stokes layer, and one induced by development of axial velocity boundary layer in the systole). In the post-exercise condition (Re=3400, α=16.7), a Lyne vortex appears in the end of systolic period and exists until the beginning of systolic phase, together with the vertices generated in the diastolic phase. Furthermore multiple period solutions were found numerically for several flow conditions. Our calculation indicates the aortic flow may have multiple period solutions, but its physiological significance is not yet clear.

Oxygen transport to a vessel of an aortic arch was numerically analyzed using the finite volume method. Oxygen delivery to the vessel is achieved through two pathways ; one is direct transport from blood flow in the lumen and the other is via the vaso vasorum. The oxygen transport from blood to the vessel consists of several processes ; release from hemoglobin, convection to the vessel wall, simple diffusion, and metabolism in the vessel. The blood is assumed to be fully developed and a flow field previously calculated from physiological aortic flow was used for the calculation of concentration fields. Our results show that even under normal physiological conditions, there exists very low O2 tension region in the middle of the vessel tissue. The secondary flow causes to make the minimum of oxygen tension near the inner side of bend. The tension near the inside is 20% less than that of the outside. This nonuniformity of oxygen tension in the circumferential direction may be associated with the localized formation of atherosclerotic lesions.

Hydrophobic mutants of the Bradyrhizohium japonicum strain 138NR were obtained by transposon Tn5 mutagenesis followed by replica-plating on polystyrene plates. Fifteen mutants were isolated at a frequency of 106. Gel-filtration analysis of the exopolysaccharides revealed that the hydrophobic mutants produced a significantly smaller amount of low-molecular-weight polysaccharides than the parent. Four of the isolated mutants formed a larger number of nodules on soybean (Glycine max L. Merr. cv. Tamahomare) than the parent, two were superior in symbiotic nitrogen fixation, and two were symbiotically defective on soybean. Competitive nodulation abilities of the mutants were examined by inoculating them to soybean with B. japonicum strain 123ET as a competitor and determining the nodule occupancy based on the antibiotic resistance. All the mutants tested except for the symbiotically defective ones were superior in their competitive nodulation ability to the parent strain. When inoculated 24 h before t...

Fully developed oscillatory flow in curved tubes is numerically solved by SIMPLER method. The calculation was carried out for Womersley number a=18, Peak Dean number x=370, and the curvature ratio δ=1/2, 1/3, 1/7. The velocity profile obtained in this study shows an inertia dominant flow due to fairly high α, however secondary flow of Lyne type does not appear for curvature ratio. It should be noted that large curvature effect induces a strong secondary flow, although the effect of curvature on the axial velocity is minor.

Arginine vasopressin (AVP) attracted attention as a potentially important neurohormonal mediator of the heart failure (HF) syndrome and hyponatremic states in humans because AVP influences renal handling of free water, vasoconstriction and myocyte biology through activation of V 2 and V 1a receptors. Current research is exploring V 2 - and dual V 1a /V 2 receptor antagonism for the treatment of hyponatremia, as well as for the congestion and edema associated with chronic HF, because vasopressin receptor antagonists might offer benefits in comparison with conventional loop diuretics. The purpose of this review is to update the current status of experimental and clinical studies with available vasopressin receptor antagonists (conivaptan and tolvaptan) and their potential role in the treatment of HF and hyponatremia of multiple causes.

Cell surface hydrophobicity of Bradyrhizobium japonicum strains 123ET and 138NR was studied as a factor in competition for the attachment to root and the nodulation of soybean. Hydrophobicity of the strains, which was measured by a biphasic water-hydrocarbon system, significantly changed with culture age having a maximum value in mid-log phase. Three days-old soybean (Glycine max cv. Tamahomare) were inoculated with the mixture of two strains, and grown for 3 weeks in sterilized vermiculite tubes. Bradyrhizobia attached to the seedling roots were released at 90 min after the inoculation by sonication and enumerated by plate counting. Two strains were distinguished by the difference in antibiotic resistance. Nodule occupancies by the strains were determined by the ratio of the strains on the seedling root surface, not in the inocula. Logarithm of the ratio of the strains on the root was highly correlated with the difference in hydrophobicity of the strains.

Gd(3+)-functionalized near-infrared emitting quantum dots were synthesized as a dual modal contrast agent for in vivo fluorescence imaging and magnetic resonance imaging.

A capillary gas chromatography-mass spectrometry for analysis of 15N-labeled amino acids and amides is described. The method is based on direct silylation of amino acids and amides with MTBSTFA and the formation of the TBDMS derivatives. The method was possible simultaneously to measure the 15N abundance ratio of amino-N and amide-N of amides, as to analysis of amino acids.

We review recent evidence which suggests that leukocytes in the circulation and in the tissue may readily respond to physiological levels of fluid shear stress in the range between about 1 and 10 dyn/cm 2, a range that is below the level to achieve a significant passive, viscoelastic response. The response of activated neutrophilic leukocytes to fluid shear consists of a rapid retraction of lamellipodia with membrane detachment from integrin binding sites. In contrast, a subgroup of non-activated neutrophils may project pseudopods after exposure to fluid shear stress. The evidence suggests that G-protein coupled receptor downregulation by fluid shear with concomitant downregulation of Rac-related small GTPases and depolymerization of F-actin serves to retract the lamellipodia in conjunction with proteolytic cleavage of beta 2 integrin to facilitate membrane detachment. Furthermore, there exists a mechanism to up- and down-regulate the fluid shear-response, which involves nitric oxide and the second messenger cyclic guanosine monophosphate (cGMP). Many physiological activities of circulating leukocytes are under the influence of fluid shear stress, including transendothelial migration of lymphocytes. We describe a disease model with chronic hypertension that suffers from an attenuated fluid shear-response with far reaching implications for microvascular blood flow.

Blood flow regulation in the cerebral microvasculature with an arcadal network was investigated using a numerical simulation. A mathematical model for blood flow in the arcadal network, based on in vivo data of cat cerebral microvasculature and flow velocity was developed. The network model consists of 45 vessel segments and 25 branching points. To simulate microvascular response to blood flow, non-reactive (solid), cerebral arteriole-like, or skeletal muscle arteriole-like responses to wall shear stress were taken into account. Numerical calculation was carried out in the flow condition where the inlet (arterial) pressure was changed from 60 to 120 mmHg. Flow-rate in each efferent vessel and the mean flow-rate over all efferent vessels were evaluated for assessment of blood supply to the local area of cerebral tissue. The simulation demonstrated the wall shear stress-induced vasodilation in the arcadal network worked to maintain the blood flow at a constant level with pressure variable in a wide range. It is suggested that an individual microvessel (segment) should join in the regulatory process of flow, interacting with other microvessels (cooperative regulation).

Flow disturbance due to microembolism in the cerebral microvasculature with an arcadal network was studied by a numerical simulation. A mathematical model for flow in the arcadal network was developed, based on in vivo data of cat cerebral microvasculature and flow velocity. The network model consisted of 45 vessel segments, and 25 branching points. To simulate microvascular responses to blood flow, the following three types of responses to wall shear stress were considered; non-reactive (solid-like), cerebral arteriole, and skeletal muscle arteriole-like responses. The numerical calculation was carried out in the condition where a feeding arteriole was occlused. Flow changes in efferent vessels were evaluated for assessment of blood supply to the local area of cerebral tissue. The present simulation has demonstrated that blood flow in efferent vessels was influenced by the topology of the vascular network and the response pattern in single vessels. The arcadal structure of arterioles might be most effective in response to flow disturbances in efferent vessels.

Autologus veins have been used clinically as a bypass conduit for reconstruction of small arteries, but there are few data available for microvascular response to arteriovenous (AV) shunting. This study was aimed to evaluate microvascular hemodynamic changes induced by creating AV anastomosis in rat hindlimb. Using intravital fluorescence videomicroscopy, we measured velocities of red blood cells (RBCs) flowing in the microvascular network in the control state, in the occlusion state where the superficial femoral artery (SFA) was occluded, and in the AV shunting state where the AV anastomosis was opened after occlusion of SFA. RBC velocities were measured in 155 capillaries of 6 rats using a dual window method and a frame-by-frame technique. The mean velocity and the coefficient of variation were 0.61 mm/sec and 0.90 in the control state, 0.34 mm/sec and 1.30 in the occlusion state, 0.83 mm/sec and 1.24 in the AV shunting state, respectively. These indicated that hemodynamic heterogeneity among capillaries increased with decrease in mean velocity following the arterial occlusion, while the AV shunting augmented the heterogeneity with increase in mean velocity. Capillaries with low perfusion (0.1 mm/sec) or high perfusion (1.0 mm/sec) were 5.8% or 20.6%, 29.6 or 5.2%, and 22.6 or 30.3% out of all measured capillaries in the control, occlusion and AV shunting conditions, respectively. In conclusion, AV shunting increased capillary perfusion and also its spatial heterogeneity, preferentially inducing high velocity in the microvasculature.

Vascular remodeling induced in rat limb by arteriovenous (AV) shunting was investigated by evaluating changes in vascular diameter and cell morphology. In Wistar rats, a vein graft was implanted in situ in the hind limb. Flow-rate in the grafted vein was assessed by measuring flow in the common femoral artery using an ultrasonic flowmeter. Nuclei and actin filaments of the venous wall were stained with propidium iodine and phalloidine-FITC, and the samples were observed using confocal laser microscopy. The grafted veins became circular in cross-section with increase in diameter during two weeks after AV shunting. Owing to the increase in diameter, the estimated wall shear stress was not increased so much as the flow-rate. The confocal laser microscopic observation showed that endothelial cells (ECs) and smooth muscle cells (SMCs) in the grafted veins were either aligned well (2 out of 8 samples), or ECs were denudated and SMCs were disrupted (in 6 out of 8 samples). The cell density of ECs was unchanged from the control level. In conclusion, the grafted vein was remodeled with morphological changes in ECs and SMCs during 2 weeks after AV shunting.

Capillary angiogenesis and remodeling induced by arteriovenous (AV) shunting in rat hind limb was investigated by evaluating changes in capillary density and diameter in the skeletal muscle subject to retrograde flow and high pressure. Wistar rats were used, and an AV anastomosis was created in the hind limb. Two weeks after AV shunting, the microvasculature in the limb was visualized by GS-lectine, and the samples were observed using confocal laser microscopy. The capillary density were increased by approximately 150% for small vessels (13 microm in diameter) under retrograde flow condition, but no change appeared for large vessels (13 microm in diameter). The capillary diameters were not significantly different between control and chronic condition. In conclusion, retrograde flow produced by AV shunting increased capillary density but it did not change the capillary diameter significantly.

Leukocyte de-activation in response to a mechanical stimulus may be an important mechanism to reduce inflammation in the circulation and cardiovascular complications. We examine here a specific form of leukocyte activation in the form of pseudopod projection, a process that is important during cell spreading and migration, but if it occurs in circulating leukocytes, may also lead to their entrapment in the microvascular network. Fresh neutrophils were activated with fMLP, suspended without adhesion to endothelium, and sheared in a cone-and-plate device while both shear stress and shear rate were measured. A fraction of the activated neutrophils retracted their pseudopods under the influence of fluid shear and returned to round shape. Pseudopod retraction was observed only in the presence of erythrocytes (at shear stresses up to approximately 25 dyn/cm(2)). At a constant hematocrit and increasing plasma viscosities with addition of macromolecules, the number of de-activated neutrophils scaled with shear stress and less so with shear rate. We examined a biochemical and rheological role of erythrocytes during shear de-activation of neutrophils. Addition of superoxide dismutase (SOD) in phosphate buffer served to enhance neutrophil de-activation by fluid shear. Replacement of erythrocytes by solid microspheres (5.4 mum) to simulate the particle properties of the erythrocytes, did not serve to enhance neutrophil de-activation unless in the presence of SOD. At higher shear stresses without erythrocytes (38-77 dyn/cm(2)), we also observed neutrophil de-activation but only in the presence of SOD. These results suggest that erythrocytes play an important role in neutrophil de-activation by reducing the superoxide level in plasma. Shear stress, rather than shear rate, is the key determinant that regulates neutrophil de-activation.

Biomathematical models for cat cerebral arteriolar network were developed for numerical evaluation of the significance of arcadal structure in the cerebral microvascular hemodynamics. Heterogeneous distribution of hematocrit and mutual co-operation in the flow regulation in the arcadal network were demonstrated using the numerical simulation.

A new nitric oxide (NO) selective electrode was developed. The tip diameter of the NO electrode is less than 10 μm. A working and a reference electrodes are located coaxially at the tip of the NO electrode. A stable NO donor SNAP (S-nitroso-N-acetyl-DL-penicillamine) and a putative NO scavenger hemoglobin (Hb) was used to examine the response of the electrode. Considering the instantaneous concentration of NO secretion previously reported in biological systems, 0.1, 1 μM NO solution was used to calibrate the electrode. From the result of the test of the electrode with SNAP and Hb, we confirmed this microcoaxial NO electrode can detect NO released from SNAP directly and continuously. From the result of calibration of the electrode, good linearity was examined in sub-molar concentration range. It is expected to detect local NO concentration in biological systems with a high space resolution.

A bacterial strain, Curtobacterium sp., isolated from a soil with zinc added possessed ω-cyclohexyl fatty acids. ω-Cyclohexyl undecanoic acid made up 47% of the total fatty acids; it was the most abundant fatty acid in the strain grown in tryptone medium. 12-Methyl tetradecanoic acid (23%) and 14-methyl hexadecanoic acid (22%) were also major fatty acids. The proportion of ω-cyclohexyl undecanoic acid increased as the pH of the medium decreased and as the culture temperature increased.

The relationship between the hydrogen uptake ability of rhizobia and their survival in soil was examined in two kinds of experiments. Indigenous rhizobia in soil were separated by a centrifugation-sonication technique into two populations differing in their location in the soil; the inner and outer areas of the soil aggregate. Each fraction was inoculated to soybean, and the nodules formed on the plants were assayed for acetylene reduction and hydrogen evolution by gas chromatography. Relative efficiency (RE) in nitrogen fixation of each nodule was assumed to be an indication of the hydrogen uptake ability of the rhizobia which formed the nodule. When the soils were incubated at 30°C for 14 days at 60% WHC moisture content, the inner fraction showed a higher ratio in the frequency of rhizobia with a RE value above 0.8 than the outer fraction, after compared to before soil incubation, respectively. Eight strains of antibiotic-resistant Bradyrhizobium japonicum, which showed different RE values, were separa...

A personal computer was used in the measurement of 15N abundance of amino acids by GC-MS. The analogue output of MS was transformed to a digital signal by an A/D converter and recorded on a hard disk of a personal computer. The ion strength ratio of the mass fragments was obtained by the manipulation of the data on the CRT.The 15N abundance of authentic amino acids showed a consistently low coefficient of variation of 0.03%. The 15N labeling ratios in free and protein-derived amino acids from Anabaena 7120 grown on 15N enriched potassium nitrate were measured using this method. The values determined had a lower 15N abundance than that of the administered 15N labeled potassium nitrate. This was most likely due to concurrent nitrogen fixation under these particular growth conditions.This method was effective in its accuracy and resulted in reduced the labor and the cost.

Effect of zinc on bacterial flora in soil was studied. Cellular fatty acid composition was analyzed with bacteria isolated from a soil incubated with glucose (C source) and ammonium sulfate (N source) in the presence or absence (control) of zinc chloride (1, 000ppm as Zn). Isolated strains were classified by the cluster analysis based on the correlation coefficient (r) computed using their fatty acid composition. At the 0.95r level strains isolated from the control soil were separated into 8 clusters and 2 of them predominated. On the other hand, isolated strains from the soil with zinc added similarly fell into 8 clusters and 4 of them became predominant successively in the incubation. Most of the strains belonging to the clusters obtained in the soil with zinc were zinc-tolerant and had low similarity to those in the control soil.

ω-Cyclohexyl undecanoic acid, an unusual fatty acid, was found in a bacterium isolated from a soil with added zinc. The ω-cyclohexyl fatty acid accounted for 46% of total fatty acids and was the most abundant fatty acid in bacteria grown in T medium. In addition, 12-methyl tetradecanoic acid (27%) and 14-methyl hexadecanoic acid (23%) were also quantitatively major fatty acids. The bacteria grew almost normally in the Zn-enriched medium, and their ω-cyclohexyl undecanoic acid content increased with Zn concentration. The results suggest that the ω-cyclohexyl fatty acid may be related to the Zn tolerance of this strain.

Discharge occur when a charged sheet is separated from a grounded metal cylinder. The discharge is named “separating discharge”. Photographs of the discharges are taken with image intensifiers aided VTR camera, powder figures of the discharges are mede, and electrical magnitude of the discharges is measured. It is found that separating discharges initiate when surface charge density σ of the electrified sheet exceeds 34 μ C/m 2 : this threshold value is independent of sheet thickness and the polarity of electrification. Length of the discharge figures decreases as σ increases to hundred or some hundred μ C/m 2 . When σ increases further, the discharges take place twice: weaker discharges at short gap point and stronger ones at large gap point. These characteristics can be explained with a partial capacitance model and with charge neutralization by preceding discharges.