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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.

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.

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.

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.

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.

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.

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.

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.

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.