Search

Double-strand breaks (DSBs) of giant DNA molecules after exposure to 1.0 MHz pulsed-wave ultrasound were quantitatively evaluated by single-molecule observation of giant DNA (T4 GT7 DNA; 166 kbp) through fluorescence microscopy. Aqueous solutions of DNA were exposed to ultrasonic waves with different sound pressures, repetition periods (1, 2, 5 ms), and pulse durations (5, 10, 50 μs). Below a threshold value of sound pressure, almost no double-strand breaks were generated, and above the threshold, the degree of damage increased in an accelerated manner as the pressure increased. DNA damage was much more severe for exposure to ultrasound with a shorter pulse duration. In addition, a longer pulse repetition period caused worse damage in DNA molecules. The effect of microbubbles on the damage induced by exposure to ultrasound had also been studied. While a result showed that a very small amount of microbubbles increased DSBs of DNA, this effect of microbubbles only weakly depended on their concentrations.

The LHCf experiment is designed to provide precise measurements of very forward neutral particle production from high energy proton-proton, proton-ion and ion-ion collisions. This information is necessary to test and tune hadronic interaction models used by ground-based cosmic rays experiments to extract the average composition of Ultra High Energy Cosmic Rays. In order to reach this goal, LHCf makes use of two small sampling calorimeters installed in the LHC tunnel at $\pm 140$ m from Interaction Point 1, both able to detect neutral particles having pseudo-rapidity $\eta > 8.4$. In LHC Run I and II, the LHCf experiment acquired data relative to p-p collisions at $\sqrt{s} = $ 0.9, 2.76, 7 and 13 TeV, and p-Pb collisions at $\sqrt{s_{NN}} = $ 5.02 and 8.16 TeV. Forward production from p-p and p-Pb collisions are not directly applicable to the tuning of the model used to simulate extensive air showers, since the first interaction between a cosmic ray and an atmospheric nucleus generally involves a light nucleus, like N or O. In LHC Run III, we will have the unique opportunity to directly measure forward production from high energy p-O collisions, without the need to obtain this information by interpolating the measurements from p-p and p-Pb collisions. In this contribution, we discuss the importance of such a measurement, focusing on all the benefits in terms of a more direct and complete input for model tuning, and on the operation plans, including the importance to take data both from high energy p-O and O-O collisions.

Purpose: Effective methods to ameliorate radiation enteropathy have not been developed. To address this issue, we investigated the reduced form of coenzyme Q10 (rCoQ10) as a potential radioprotector in a mouse model. Methods and Materials: rCoQ10 was added to a standard laboratory mouse diet at a final concentration of 1.0% 9 days before irradiation and 30 days thereafter or dissolved in corn oil and administered transorally. Accumulated amounts of coenzyme Q10 (CoQ10) or coenzyme Q9 in the intestine were measured by high-performance liquid chromatography. Reactive oxygen species (ROS), apoptosis, and morphologic changes in the intestine were assessed by immunohistochemistry after administration of 13 Gy of x-ray to the mouse abdomen. Body weight and survival were monitored for 30 days after irradiation. Cytotoxicity using 3 human cancer cell lines and the tumor growth-inhibiting effect in a xenograft were investigated to determine whether rCoQ10 interferes with radiation-specific cytotoxic effects on tumor growth. Results: CoQ10 was greatly accumulated in all sections of the intestine after both massive transoral dosing and dietary administration, whereas coenzyme Q9 was not. Administration of rCoQ10 suppressed ROS production and inhibited apoptosis in the crypts, resulting in preservation of villi structures after irradiation. Notably, 92% of mice fed the rCoQ10-supplemented diet were healthy and alive 30 days after irradiation, whereas 50% of control mice died (P < .05). Moreover, rCoQ10 did not interfere with radiation-specific cytotoxic effects on tumors either in vitro or in vivo. Conclusions: Administration of rCoQ10 led to its accumulation in the intestine and induced radioprotective effects by inhibiting ROS-mediated apoptosis, thereby preserving intestinal structures. Our results indicated that rCoQ10 supplementation effectively ameliorated radiation enteropathy.

The development of potentially safe radiosensitizing agents is essential to enhance the treatment outcomes of radioresistant cancers. The titanium peroxide nanoparticle (TiOxNP) was originally produced using the titanium dioxide nanoparticle, and it showed excellent reactive oxygen species (ROS) generation in response to ionizing radiation. Surface coating the TiOxNPs with polyacrylic acid (PAA) showed low toxicity to the living body and excellent radiosensitizing effect on cancer cells. Herein, we evaluated the mechanism of radiosensitization by PAA-TiOxNPs in comparison with gold nanoparticles (AuNPs) which represent high-atomic-number nanoparticles that show a radiosensitizing effect through the emission of secondary electrons. The anticancer effects of both nanoparticles were compared by induction of apoptosis, colony-forming assay, and the inhibition of tumor growth. PAA-TiOxNPs showed a significantly more radiosensitizing effect than that of AuNPs. A comparison of the types and amounts of ROS generated showed that hydrogen peroxide generation by PAA-TiOxNPs was the major factor that contributed to the nanoparticle radiosensitization. Importantly, PAA-TiOxNPs were generally nontoxic to healthy mice and caused no histological abnormalities in the liver, kidney, lung, and heart tissues.

Purpose/Objectives: The mechanism of intercellular communication after radiation exposure in cancer cells remains fully undetermined. Exosomes are lipid bilayer-constituted, membrane-enclosed small vesicles that are recognized as mediators transporting a variety of intracellular components including miRNA. Here we identified the novel role of exosomes released from irradiated cells to neighboring cancer cells. Materials/Methods: Human pancreatic cancer cell line MIAPaCa-2 was used in this study. Purified exosome product (PEP) was obtained from cultured media by ultra-centrifugation. PEP was morphologically confirmed by transmission electron microscopy (TEM), and analyzed by NanoSight. Exosome-specific surface markers CD9 and CD63 were evaluated by western blotting. Endocytosis of irradiated exosomes was confirmed by fluorescent microscopy by using the PKH26 dye. Cell survival after irradiation was evaluated by a colony-forming assay. Intracellular reactive oxygen species (ROS) levels were determined using the oxidation-sensitive fluorescent probe dye C-H 2 DCF, and DNA damage was evaluated by detecting phosphorylated histone 2AX (γ-H2AX) foci by immunocytochemistry. MiRNAs were isolated from the exosomes after 5 Gy or 8 Gy of irradiation and comprehensive miRNA expression analysis was performed by miRNA microarray analyses. Expressions of Cu/Zn superoxide dismutase enzyme (SOD1) or Mn superoxide dismutase enzyme (SOD2), catalase, and glutathione peroxidase were studied to determine whether the exosomes received by the neighboring cells may have influence to them and lead to production of ROS or not. Results: Exosome characteristics were confirmed by multiple methods. The uptake of irradiated exosomes was significantly higher than that of nonirradiated exosomes. Notably, nonirradiated neighboring cells with irradiated exosomes induced higher intracellular levels of ROS, and a higher frequency of DNA damage. These neighboring cells also showed greater sensitivity to radiation. Seven upregulated and 5 downregulated miRNAs were identified that correlated with the miRNA microarray analyses results obtained after 5 Gy and 8 Gy radiation. Among them, miR-6823-5p was identified as a possible candidate for SOD1 inhibition, leading to intracellular ROS production and DNA damage. Conclusions : This is the first study to determine that irradiated exosomes can enhance the radiation effect via ROS production in cancer cells. This novel finding may lead to the understanding of the bystander effect of neighboring cancer cells.

Purpose To develop a novel biocompatible solid fiducial marker that prevents radiopaque imaging artifacts and also maintains high imaging contrast for kilovoltage x-ray image-guided radiation therapy. Methods The fiducial marker was made of pure zinc. An in-house water-equivalent phantom was designed to evaluate artifacts and visibility under various simulated treatment scenarios. Image artifacts were quantitatively assessed in terms of the metal artifact index (MAI) on kilovoltage computed tomography (CT) and cone-beam CT (CBCT) scans. Marker visibility was evaluated on two types of kilovoltage planar x-ray images in terms of the contrast-to-background ratio (CBR). Comparisons with a conventional gold fiducial marker were conducted. Results The use of zinc rather than a gold marker mitigates imaging artifacts. The MAI near the zinc marker decreased by 76, 79, and 77 % in CT, and by 77 (81), 74 (80), and 79 (85) % in CBCT full-fan (half-fan) scans, when using one-, two-, and three-marker phantom settings, respectively. The high-contrast part of the zinc marker exhibited CBRs above 2.00 for 28/32 exposures under four (lung, tissue, low-density bone, and high-density bone) different simulation scenarios, making its visibility comparable to that of the gold marker (30/32 exposures with CBRs > 2.00). Conclusions We developed a biocompatible, artifact-robust, and highly visible solid zinc fiducial marker. Although further evaluation is needed in clinical settings, our findings suggest its feasibility and benefits for kilovoltage x-ray image-guided radiation therapy.

Excess energy intake causes obesity, which leads to insulin resistance and various other complications of metabolic syndrome, including diabetes, atherosclerosis, dyslipidemia, and nonalcoholic fatty liver disease. Although recent studies have depicted altered lipid metabolism as an underlying feature, the detailed mechanisms are still unclear. Here we describe a possible role in high-fat diet (HFD)-induced obesity for monoacylglycerol lipase (MGL), an enzyme that is also known to hydrolyze the endocannabinoid 2-arachidonoylglycerol in brain. MGL-deficient [MGL-knockout (KO)] mice fed a HFD gained less body weight than wild-type mice and were protected from insulin resistance and hepatic steatosis. Food intake and energy expenditure were not altered in MGL-KO mice, but blood triglyceride levels after oral olive oil gavage were suppressed, indicating a role for MGL in intestinal fat absorption. Experiments with cannabinoid receptor type 1 (CB1)/MGL double-KO mice revealed that these phenotypes may include mechanisms that are independent of CB1-receptor-mediated endocannabinoid functions. We also noted that MGL-KO mice had less preference for HFD over normal chow diet. Oral but not intraperitoneal lipid administration strongly suppressed the appetites of MGL-KO and CB1/MGL double-KO mice, but not of wild-type and CB1-KO mice. Appetite suppression was reversed by vagotomy, suggesting involvement of MGL in the gut-brain axis regulation of appetite. Our results provide mechanistic insights of MGL's role in diet-induced obesity, lipid metabolic disorder, and regulation of appetite.-Yoshida, K., Kita, Y., Tokuoka, S. M., Hamano, F., Yamazaki, M., Sakimura, K., Kano, M., Shimizu, T. Monoacylglycerol lipase deficiency affects diet-induced obesity, fat absorption, and feeding behavior in CB1 cannabinoid receptor-deficient mice.

Microbubbles shelled with soft materials are expected to find applications as ultrasound-sensitive drug delivery systems, including through sonoporation. Microbubbles with specific vibrational characteristics and long intravascular persistence are required for clinical uses. To achieve this aim, the kinetics of the microbubble shell components at the gas/liquid interface while being subjected to ultrasound need to be better understood. This paper investigates the vibration characteristics and lifetime of single microbubbles coated with a poloxamer surfactant, Pluronic F-68, and 1,2-dimyristoyl

Increased skin and subcutaneous tissue stiffness in patients with early-stage lymphedema has been reported. The purpose of this study was to examine the use of shear wave elastography (SWE) for evaluating lower extremity lymphedema (LEL).For 10 lower extremities of normal controls and 72 limbs of patients with gynecological cancer whose lymphatic function was categorized into six stages based on the range of dermal backflow (DBF) observed in indocyanine green (ICG) lymphography, SWE was performed and shear wave velocity (SWV) of the dermis and three layers of subcutaneous tissue at the thigh and calf were recorded. Twenty-five patients underwent thigh tissue histological and dermal thickness examinations.The strongest correlation between the ICG DBF stage and SWV during SWE was observed on the dermal layer of the thigh (p0.01, R = 0.67). There was a significant correlation between the dermal thickness of the thigh and the ICG DBF stage (p0.01, R = 0.87) and also between the dermal thickness of the thigh and SWV (p0.01, R = 0.73).Noninvasive, objective evaluation of LEL severity using SWE was well correlated with lymphatic function as determined by ICG lymphography. The DBF changes in the dermis of the thigh best reflected the changes in lymphatic function. Dermal thickness variations may partially account for differences in SWV.

We investigated the effects of small-scale oscillation (SSO) on toxic Microcystis cells. The oscillating device was made of silicon with two axes that had a diameter of ∼40 mm, and a frequency of 2.5 Hz was observed at 150 rpm. The SSO was effective in inhibiting Microcystis growth. Microcystin release was not observed, whereas cell density barely increased in the oscillating group. Cell size and morphology of the oscillating group were no different from the control group. However, cell quotas of chl.a and microcystin in the oscillating group were half the level of the control group. Crucially, a number of large-sized holes were observed and layered long linear thylakoids were rarely observed in the oscillating group. Therefore, SSO was found to be very effective in Microcystis growth inhibition, and it caused ultrastructural changes without damage to the cell membrane and subsequent microcystin release.

Lipid-coated microbubbles (MBs) with an indocyanine green (ICG) derivative were fabricated for ultrasound and near-infrared (NIR) fluorescence dual imaging. We characterized the NIR-fluorescence intensity, stability and viscoelastic properties of the encapsulating lipid shell, focusing on the influence of the ICG derivative and lipid compositions. In terms of the NIR fluorescence intensity, the fluorescence intensity of the MBs (with the ICG derivative) was significantly affected by the lipid composition of the MB shell. Regarding the contrast agent used for ultrasound imaging, the stability of the MBs and viscoelastic properties of shell also depended on the lipid compositions, while the incorporation of the ICG derivative into the MB shells had a negligible effect. The performance of this contrast agent for ultrasound and NIR fluorescence dual-imaging exhibited a significant trade-off relationship for the lipid composition.

In previous studies, the double-Nakagami (DN) model has been proposed for fatty liver assessment and applied to in vivo rat livers and clinical data sets. The healthy liver structure filter (HLSF) method, which extracts non-healthy areas using two DN parameters, has also been proposed. In this paper, we first verify the accuracy of the DN model and the HLSF method for acoustic fields at 15 and 5 MHz, which were reproduced using numerical simulation. We then apply the method to clinical data sets of livers observed using a frequency of 3 MHz and investigate the method’s clinical usefulness. A positive correlation ( r = 0.28 ) was found between the ratio of the non-healthy area and fat mass. Although the results were inferior to the results produced using 15 MHz ultrasound ( r = 0.96 ), we found that it was possible to detect the difference between a normal liver and a fatty liver even at a lower frequency.

Diphenylarsinic acid (DPAA) was detected as the primary compound responsible for the arsenic poisoning that occurred in Kamisu, Ibaraki, Japan, where people using water from a well that was contaminated with a high level of arsenic developed neurological (mostly cerebellar) symptoms and dysregulation of regional cerebral blood flow. To understand the underlying molecular mechanism of DPAA-induced cerebellar symptoms, we focused on astrocytes, which have a brain-protective function. Incubation with 10 µM DPAA for 96 h promoted cell proliferation, increased the expression of antioxidative stress proteins (heme oxygenase-1 and heat shock protein 70), and induced the release of cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Furthermore, DPAA overpoweringly increased the phosphorylation of three major mitogen-activated protein kinases (MAPKs) (ERK1/2, p38MAPK, and SAPK/JNK), which indicated MAPK activation, and subsequently induced expression and/or phosphorylation of transcription factors (Nrf2, CREB, c-Jun, and c-Fos) in cultured rat cerebellar astrocytes. Structure-activity relationship analyses of DPAA and other related pentavalent organic arsenicals revealed that DPAA at 10 µM activated astrocytes most effective among organic arsenicals tested at the same dose. These results suggest that in a cerebellum exposed to DPAA, abnormal activation of the MAPK-transcription factor pathway and irregular secretion of these neuroactive, glioactive, and/or vasoactive cytokines in astrocytes can be the direct/indirect cause of functional abnormalities in surrounding neurons, glial cells, and vascular cells: This in turn might lead to the onset of cerebellar symptoms and disruption of cerebral blood flow.

Anti-desmoglein (Dsg) 1 and Dsg3 IgG autoantibodies in pemphigus foliaceus and pemphigus vulgaris cause blisters through loss of desmosomal adhesion. It is controversial whether blister formation is due to direct inhibition of Dsg, intracellular signaling events causing desmosome destabilization, or both. Recent studies show that heterophilic binding between Dsg and desmocollin (Dsc) is the fundamental adhesive unit of desmosomes. To eliminate cellular contributions to potential pathogenicity of pemphigus antibodies, bead assays coated with recombinant Dsg1, Dsc1, Dsg3, or Dsc3 ectodomains were developed. A mixture of Dsg beads and Dsc beads formed large aggregates, confirming that the heterophilic binding is dominant. The pathogenic anti-Dsg1 and anti-Dsg3 mAbs, which bind the transadhesive interface, blocked the aggregation of Dsg1/Dsc1 and Dsg3/Dsc3 beads, respectively, whereas nonpathogenic mAbs did not. All sera tested from eight patients with pemphigus foliaceus and eight patients with mucosal pemphigus vulgaris with active disease inhibited the adhesion of Dsg1/Dsc1 and Dsg3/Dsc3 beads, respectively. When paired sera obtained from seven patients with pemphigus foliaceus and six patients with pemphigus vulgaris in active disease and remission were compared, the former inhibited aggregation better than the latter. These findings strongly suggest that steric hindrance of heterophilic transinteraction between Dsg and Dsc is important for disease pathology in both pemphigus foliaceus and pemphigus vulgaris.

Gastrointestinal toxicity is frequently observed secondary to accidental or therapeutic radiation exposure. However, the variation in the intestinal metabolites after abdominal radiation exposure remains ambiguous. In the present study, C57BL/6 mice were exposed to 0, 2, and 20 Gy irradiation dose. The Head and chest of each mouse were covered with a lead shield before x-ray irradiation. 24 h post-irradiation treatment, intestinal tissue of each mouse was excised and prepared for metabolites measurement using gas chromatography-mass spectrometry (GC-MS). Our comprehensive analysis of metabolites in the intestinal tissues detected 44 metabolites after irradiation, including amino acids, carbohydrates, organic acids, and sugars. Amino acid levels in the intestinal tissue gradually rose, dependent on the radiation dose, perhaps as an indication of oxidative stress. Our findings raise the possibility that amino acid metabolism may be a potential target for the development of treatments to alleviate or mitigate the harmful effects of oxidative stress-related gastrointestinal toxicity due to radiation exposure., Highlights • Gastrointestinal damage frequently results from radiation exposure. • We analyzed the metabolic profile after local irradiation to the intestine. • Amino acid levels in the intestinal tissue rose dependent on the radiation dose. • Amino acid metabolism may be a good target for future therapies.

Miniature and highly sensitive biosensors are attractive. Quasi-shear mode c-axis tilted ScAIN film resonator is suitable for sensing biomolecules because they can operate in requid. Higher k'152 value of ScAIN films makes it possible to improve signal to noise ratio. In this study, the streptavidin-biotin interactions were monitored by using the c-axis tilted ScAlN film quasi-shear mode resonators.

Vanadium-based porous coordination polymers (or metal–organic frameworks) possess both porous and electronic properties, which make these new materials appealing for applications in molecular separation, sensing and heterogeneous catalysis. Their integration into systems that fully exploit their intrinsic properties requires versatile methods allowing assembly of the PCP crystals into well-defined films, patterns, fibers or the formation of heterostructures. In this contribution, polycrystalline macrostructures and heterostructures made of [V(OH)ndc]n (ndc = 1,4-naphthalenedicarboxylate) PCP crystals were synthesized through a dissolution–recrystallization process, so-called coordination replication, where a pre-shaped V2O5 sacrificial phase was replaced by well-intergrown PCP crystals in the presence of H2ndc as an organic linker and under a reductive environment. In this process, V2O5 acts both as the metal source and as the template that provides the shape to the resulting mesoscopic polycrystalline architecture. Ascorbic acid, acting as the reducing agent, both promotes the dissolution of the sacrificial V2O5 phase and provides the VIII species required for the construction of the [V(OH)ndc]n framework. Two-dimensional patterns were successfully synthesized by applying this procedure.

We investigated the encapsulation of bioactive molecules such as L-glutamic acid (Glu) into a series of porous coordination polymers (PCPs) based on aluminum hydroxy dicarboxylates [Al(OH)(L)]n (L = dicarboxylate ligand) and the molecular release therefrom. The use of 2,6-naphthalene dicarboxylate (2,6-ndc), 1,4-benzene dicarboxylate (1,4-bdc) and 1,4-naphthalene dicarboxylate (1,4-ndc) as ligands allows us to systematically tune the pore size and the flexibility of frameworks while keeping the same topology and thus to investigate the effect of those parameters upon both adsorption and release of Glu. We revealed the impact of zwitterionic nature of Glu upon loading efficiency; optimal loading pH was shown to be that for which Glu bears both positive and negative charges. Whereas the loading capacity of PCPs is governed by the pore size ([Al(OH)(2,6-ndc)]n > [Al(OH)(1,4-bdc)]n > [Al(OH)(1,4-ndc)]n), the adsorption isotherm clearly revealed that small or flexible pores induce the stronger Glu–PCP interaction. The release experiments of Glu from PCPs in a physiological media (pH = 7.4, 37 °C) demonstrated the exceptional stabilization of Glu within [Al(OH)(1,4-bdc)]n, compared to those within the other frameworks; whereas the rigid frameworks of [Al(OH)(2,6-ndc)]n and [Al(OH)(1,4-ndc)]n spontaneously released almost the entire Glu contents within 10 h, 70% of Glu loaded within [Al(OH)(1,4-bdc)]n still remained therein over 24 h. Interestingly, the burst release of Glu was triggered by increasing temperature up to 80 °C, at which the framework changed its structure from a closed phase to the open phase.

Non-invasive diagnosis of non-alcoholic steatohepatitis (NASH) can be achieved by quantitative ultrasound (QUS), which requires a detailed understanding tissue-specific acoustic microstructure at cellular scale (i.e., 10 μm). This study focuses on obtaining speed of sound (c) of organelles (i.e., nucleus, cytoplasm, and fibers) using 250-MHz ultrasound in rat livers. Three different pathological stages were investigated in rat models: normal (control: n = 2), NASH (n = 3), and fibrosis model (n = 6). After animal sacrifice, each liver was fixed in formalin and a 7-μm thin section was affixed to glass slide using a microtome. A scanning acoustic microscopy system equipped with a 250-MHz center-frequency transducer was employed to obtain two-dimensional (2D) maps of c with a spatial resolution of 7-μm. The histological images were used to detect the positions of the nuclei, cytoplasm and fibers, and the corresponding c values from obtained from the co-registered 2D c map. For all organelles, Kruskal-Wallis test reveals a significant difference (p < 0.01) between all paired stages. These results reveals that c is the highest in fibrosis livers for all organelles. Additionally, in NASH livers, c of nuclei and cytoplasm were the lowest, which could be due to intracellular fluid or ballooning of the whole cell. As expected, c in fiber changes with pathologic state. Results reveal that c of the nucleus and cytoplasm also changes with pathologic state and that we were able to carefully assess these differences using 250-MHz ultrasound. These results could prove invaluable to improve in vivo QUS investigations of livers.

Localizing tumors and securing resection margins are sometimes difficult especially during laparoscopic surgery for malignant diseases. Therefore, we have developed a tissue marker, which is composed of giant cluster-like vesicles (GCVs) encapsulating near-infrared (NIR) fluorescent liposomes and X-ray contrast agents coated with lipids. The GCVs facilitate localizing tumors preoperatively and intraoperatively because they have superior retention effect in tissue. In addition, the GCVs can work as reservoir of a fluorescent tracer for detecting sentinel lymph nodes if they are destroyed. We propose the method to assess the destruction of GCVs using ultrasound contrast agents (UCAs) and demonstrate the effect.

Recent investigations have suggested that semaphorins, which are known repulsive axon guidance molecules, may play a crucial role in maintaining brain homeostasis by regulating microglial activity. Sema3A, secreted in higher amounts from injured neurons, is considered to suppress excessive inflammatory responses by inducing microglial apoptosis through its binding to Plexin-A1 receptors on activated microglia. To clarify the in vivo role of Plexin-A1-mediated signaling in lipopolysaccharide (LPS)-induced injury in mouse brain, we examined the neuroinflammatory changes initiated by LPS administration to the cerebral ventricles of wild-type (WT) and Plexin-A1-deficient (−/−) mice. WT mice administered LPS exhibited a significantly higher expression of COX-2, iNOS, IL-1β and TNF-α in the hippocampus, and a significantly greater ventricular enlargement and intracerebral infiltration of leukocytes, as compared with the saline-treated group. By contrast, Plexin-A1−/− mice administered LPS did not exhibit a significantly increased expression of COX-2, iNOS, IL-1β or TNF-α in the hippocampus as compared with the saline-treated group. Plexin-A1−/− mice administered LPS did not show significant increases in ventricle size or infiltration of leukocytes into the brain, as compared with the saline-treated group. In WT, but not in the Plexin-A1−/− primary microglia treated with LPS, Sema3A induced significantly more nitric oxide production than in the immunoglobulin G control. These results revealed the crucial role of the Sema3A-Plexin-A1 interaction in the Toll-like receptor 4-mediated signaling of the LPS-induced activation of microglia. Thus, results of the present study revealed the essential role of Plexin-A1 in the development of LPS-induced neuroinflammation in mice, suggesting the possible application of microglial control of the semaphorin-plexin signaling system to the treatment of LPS-induced encephalopathy and other psychiatric diseases associated with neuroinflammation.

N -Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of l -2-phenylglycine to produce (2 S )-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA , was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N -acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N -acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N -acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for l -2-phenylglycine and its chloro- and hydroxyl-derivatives. The K m and V max values for l -2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min −1 · mg protein −1 , respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N -acetyltransferase and an arylalkylamine N -acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to l -2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10

Ultrasound sonification stimulates the release of pharmaceutical compounds from hydrogels. At the surface of hydrogels, cavitation, cavities formed in liquid, activates to stimulate that release under low-frequency ultrasound. Under high-frequency ultrasound, although cavitation activities are highly suppressed, the compounds are still released. Although it remains elusive how high-frequency ultrasound stimulates this release, one hypothesis is that the internal diffusivity is enhanced. In this study, internal diffusivities in agarose gels were estimated with fluorescent recovery after photobleaching (FRAP) analysis. Under 1-MHz ultrasound sonification, internal diffusivity in agarose gels was enhanced. The enhancement of internal diffusivity was larger than that with temperature elevation alone, although temperature elevation was also observed along with the ultrasound sonification. Thus, we found that high-frequency ultrasound sonification enhances internal diffusivity in agarose gels. This enhancement was, at least in part, independent of temperature elevation.

Rigorous and consistent formulations of basic equations of interfacial area transport were derived using correlation functions of the characteristic function of each phase and velocities of each phase. The turbulent transport term of interfacial area concentration was consistently derived and related to the difference between the interfacial velocity and the averaged velocity of each phase. Constitutive equations of turbulent transport terms of interfacial area concentration were proposed for bubbly flow. New transport model and constitutive equations were developed for churn flow. These models and constitutive equations are validated by experimental data of radial distributions of the interfacial area concentration in bubbly flow and churn flow.

Background Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by autoantibodies (Abs) against desmoglein 1 (Dsg1). PF sera contain polyclonal Abs which are heterogeneous mixture of both pathogenic and non-pathogenic Abs, as shown by isolation of monoclonal Abs (mAbs). Objective To investigate how pathogenic and non-pathogenic anti-Dsg1 Abs contribute to blister formation in PF. Methods Using organ-cultured human skin, we compared the effect of a single pathogenic anti-Dsg1 IgG mAb, a single non-pathogenic anti-Dsg1 IgG mAb, and their mixture on blister formation as analyzed by histology, subcellular localization of IgG deposits and desmosomal proteins by confocal microscopy, and desmosomal structure by electron microscopy. In addition, we measured keratinocyte adhesion by an in vitro dissociation assay. Results 24 h after injection, a single pathogenic anti-Dsg1 IgG caused a subcorneal blister with IgG and Dsg1 localized linearly on the cell surface of keratinocytes. A single non-pathogenic anti-Dsg1 IgG bound linearly on the keratinocytes but did not induce blisters. A pathogenic and a non-pathogenic IgG mAb injected together caused an aberrant granular pattern of IgG and Dsg1 in the lower epidermis with blister formation in the superficial epidermis. Electron microscopy demonstrated that the mixture of mAbs shortened desmosomal lengths more than a single mAb in the basal and spinous layers. Furthermore, although Dsg1 clustering required both cross-linking of Dsg1 molecules by the non-pathogenic IgG plus a pathogenic antibody, the latter could be in the form of a monovalent single chain variable fragment, suggesting that loss of trans-interaction of Dsg1 is required for clustering. Finally, a p38MAPK inhibitor blocked Dsg1 clustering. When pathogenic strength was measured by the dissociation assay, a mixture of pathogenic and non-pathogenic IgG mAbs disrupted keratinocyte adhesion more than a single pathogenic mAb. This pathogenic effect was only partially suppressed by the p38MAPK inhibitor. Conclusion These findings indicate that a polyclonal mixture of anti-Dsg1 IgG antibodies enhances pathogenic activity for blister formation associated with p38MAPK-dependent Dsg1 clustering and that not only pathogenic antibodies but also non-pathogenic antibodies coordinately contribute to blister formation in PF.

Acoustic properties of free fatty acids present in the liver were studied as a possible basis for non-invasive ultrasonic diagnosis of non-alcoholic steatohepatitis. Acoustic impedance was measured for the following types of tissue samples: Four pathologic types of mouse liver, five kinds of FFAs in solvent and five kinds of FFAs in cultured Huh-7 cells. A transducer with an 80-MHz center frequency was incorporated into a scanning acoustic microscopy system. Acoustic impedance was calculated from the amplitude of the signal reflected from the specimen surface. The Kruskal-Wallis test revealed statistically significant differences (p

To evaluate the role of intravesical prostatic protrusion (IPP) as a predictive factor for adverse clinical outcomes in patients treated with dutasteride for lower urinary tract symptoms secondary to benign prostatic enlargement (BPE).In total, 111 patients treated with dutasteride for symptomatic BPE were analyzed. Stepwise multivariate logistic regression was applied to evaluate predictors for acute urinary retention (AUR) or benign prostatic hyperplasia (BPH)-related surgery. We applied an IPP cutoff value of 10 mm. The clinical variables were assessed using univariate analysis.Of 111 patients, 27 (24.3%) developed AUR or required surgical intervention. On multivariate analysis, IPP remained as the independent predictor for AUR and need for BPH-related surgery (odds ratio, 1.27; P .001). Both international prostate symptom score and maximum urinary flow rate significantly improved in patients with low IPP (P = .03 and P .001, respectively), but not in those with high IPP. No significant reduction was found in the degree of IPP despite the significant reduction in prostate volume after dutasteride treatment (P = .84 and P .001, respectively). The 3-year cumulative incidence of AUR or BPH-related surgery in the low IPP group vs the high IPP group was 9.9% vs 71.5%, respectively (P .001).High IPP is associated with a higher risk of treatment resistance, AUR, or the need for prostatic surgery in patients receiving dutasteride treatment for symptomatic BPE. Dutasteride might not be effective for IPP reduction.

The present study deals with in silico prediction and in vitro evaluation of the selective cytotoxic effects of triterpenoids on tumorigenic human c-Ha-ras and mouse c-myc cotransfected highly metastatic serum-free mouse embryo-1 (r/m HM-SFME-1) cells. Ligand fitting of five different triterpenoids to 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) was analyzed with a molecular modeling method, and glycyrrhetinic acid (GA) was the best-fitted triterpenoid to the ligand binding site in 11βHSD2. Analysis of antiproliferative effects revealed that GA, oleanolic acid, and ursolic acid had selective toxicity against the tumor cells and that GA was the most potent triterpenoid in its selectivity. The toxic activity of the tested triterpenoids against the tumor cells showed good correlations with the partition coefficient (logP) and polar surface area values. Time-lapse microscopy, fluorescence staining, and confocal laser scanning microscopic observation revealed that GA induced morphologic changes typical of apoptosis such as cell shrinkage and blebbing and also disrupted the cytoskeletal proteins. Furthermore, GA exhibited a strong inhibitory effect on 11βHSD2 activity in the tumor cells. Our current results suggest that analysis of the ligand-receptor interaction between triterpenoids and 11βHSD2 can be utilized to predict their antitumor effects and that GA can be used as a possible chemopreventive and therapeutic antitumor agent. To the best of our knowledge, this is the first report on in silico prediction of the toxic effects of triterpenoids on tumor cells by 11βHSD2 inhibition.

Natural products from plants are expected to play significant roles in creating new, safe and improved chemopreventive and therapeutic antitumor agents. Selectivity is also an important issue in cancer prevention and therapy. The present study was designed to extend our previous study on the c-Ha-ras and c-myc-induced tumor cell-selective antiproliferative effects of a licorice component, glycyrrhetinic acid (GA). An in silico ligand-receptor docking simulation revealed that GA acts as an 11β-hydroxysteroid dehydrogenase type 2 inhibitor. GA disrupted the redox balance in tumor cells through upregulation of reactive oxygen species and downregulation of glutathione (GSH). The GA-induced GSH reduction and cytotoxicity were enhanced by an inhibitor of GSH, l-buthionine-[S,R]-sulfoximine. N-acetyl-l-cysteine, an antioxidant and precursor of GSH, restored the GA-induced GSH reduction and cytotoxicity in tumor cells. Taken together, these data highlighting the downregulation of GSH by GA and the efficacy of GSH in ameliorating GA-mediated cytotoxicity support the notion that GSH is involved in the selective toxicity of GA toward tumor cells.

Hollow microcapsules have been considered for potential applications as drug or gene carriers. This paper describes an investigation into the mechanical properties of microcapsules with a biocompatible polylactic acid (PLA) shell that can be destroyed using ultrasound irradiation. The microcapsules had a radius of 1 to 25 μm and a shell thickness of 100 nm to 3 μm, and their response to ultrasound pulses with a center frequency of 700 kHz to 2 MHz was investigated. It was found that approximately 50% of capsules with a radius of 20 μm were destroyed using pulses with a pressure amplitude of 50 kPa and a frequency of 700 kHz, which is close to the resonance frequency of the capsules.

To evaluate the neck size of ordered cage-type mesoporous silica FDU-12 and also to elucidate the origin of the gradual desorption always observed for the ordered silicas with cage-like pores of large necks, we measured successive adsorption of water at 283 K and nitrogen or argon at 77 K on five kinds of FDU-12 samples. For all the materials examined here, the amount of nitrogen or argon condensed inside the large cavities decreased rapidly over a relatively small range of water filling with an increase in water filling. Eventually, it reached a small value at water filling, well before the onset of pore condensation of water inside the large cavities. This clearly indicates that most of the cavities are isolated from bulk nitrogen or argon gas with water frozen in the necks. With the assumption that the necks in cage-like pores are cylindrical in shape, we assessed the pore size distribution of the necks available for desorption from the equilibrium relative pressure of water, in which the amount of nit...

Podoplanin is distinctively overexpressed in oral squamous cell carcinoma than oral benign neoplasms and plays a crucial role in the pathogenesis and metastasis of oral squamous cell carcinoma but its diagnostic application is quite limited. Here, we report a new near-infrared fluorescence imaging method using an indocyanine green (ICG)-labeled anti-podoplanin antibody and a desktop/a handheld ICG detection device for the visualization of oral squamous cell carcinoma-xenografted tumors in nude mice. Both near-infrared imaging methods using a desktop (in vivo imaging system: IVIS) and a handheld device (photodynamic eye: PDE) successfully detected oral squamous cell carcinoma tumors in nude mice in a podoplanin expression-dependent manner with comparable sensitivity. Of these 2 devices, only near-infrared imaging methods using a handheld device visualized oral squamous cell carcinoma xenografts in mice in real time. Furthermore, near-infrared imaging methods using the handheld device (PDE) could detect smaller podoplanin-positive oral squamous cell carcinoma tumors than a non-near-infrared, autofluorescence-based imaging method. Based on these results, a near-infrared imaging method using an ICG-labeled anti-podoplanin antibody and a handheld detection device (PDE) allows the sensitive, semiquantitative, and real-time imaging of oral squamous cell carcinoma tumors and therefore represents a useful tool for the detection and subsequent monitoring of malignant oral neoplasms in both preclinical and some clinical settings.

Major histocompatibility complex (MHC) class II alleles HLA-DQ8 and the mouse homologue I-A(g7) lacking a canonical aspartic acid residue at position beta57 are associated with coeliac disease and type I diabetes. However, the role of this single polymorphism in disease initiation and progression remains poorly understood. The lack of Asp 57 creates a positively charged P9 pocket, which confers a preference for negatively charged peptides. Gluten lacks such peptides, but tissue transglutaminase (TG2) introduces negatively charged residues at defined positions into gluten T-cell epitopes by deamidating specific glutamine residues on the basis of their spacing to proline residues. The commonly accepted model, proposing that HLA-DQ8 simply favours binding of negatively charged peptides, does not take into account the fact that TG2 requires inflammation for activation and that T-cell responses against native gluten peptides are found, particularly in children. Here we show that beta57 polymorphism promotes the recruitment of T-cell receptors bearing a negative signature charge in the complementary determining region 3beta (CDR3beta) during the response against native gluten peptides presented by HLA-DQ8 in coeliac disease. These T cells showed a crossreactive and heteroclitic (stronger) response to deamidated gluten peptides. Furthermore, gluten peptide deamidation extended the T-cell-receptor repertoire by relieving the requirement for a charged residue in CDR3beta. Thus, the lack of a negative charge at position beta57 in MHC class II was met by negatively charged residues in the T-cell receptor or in the peptide, the combination of which might explain the role of HLA-DQ8 in amplifying the T-cell response against dietary gluten.

We propose a novel contrast echo imaging method using crossed beams of two different ultrasonic frequencies as an ultrasound method of diagnostic cancer, which we call the crossed beam contrast echo (CBCE) method. The sum or difference frequency components that are derived from the nonlinear oscillation of the microbubbles driven by dual-frequency ultrasound are used in the CBCE method. The blood flow information can only be obtained in the area of the crossed beams, because no sum or difference frequency components are generated by the propagation of biological soft tissues except in the crossed beam area. We discussed the driving conditions under which the sum frequency component is most efficiently generated, and experimentally confirmed the area in which the sum frequency component is generated. As a result, the sum frequency component was larger than the other harmonic components when its frequency was equal to the resonance frequency of the microbubbles. It was confirmed that the area in which the large sum frequency component is generated was equal to the crossing area of the two beams.

Early diagnosis of non-alcoholic steatohepatitis (NASH) is highly desired because NASH can lead to cirrhosis or even to hepatocellular carcinoma in some severe cases. Towards non-invasive diagnosis with ultrasound, we studied free fatty acids (FFAs) present in the liver, which is the organ most likely to be affected by the disease. As a preclinical study, we performed acoustic-impedance measurements of five kinds of FFAs in solvent or in cultured Huh7 cells. To measure the acoustic impedance, a concave transducer with an 80-MHz center frequency was incorporated in a scanning acoustic microscopy system. One-way ANOVA showed statistically-significant differences (p < 0.05) in acoustic impedance among the FFAs in FFA solvent and with cultured Huh7 cells. These results suggest that each of the FFAs, especially PA, OA and PAOA could be distinguished from each other regardless of whether they were in solution or in absorbed by cells.

Bi films that we fabricated with vacuum-evaporation deposition show a crystal structure very similar to that of bulk Bi. However they have granular structure on sub-micron scales, and the electrical resistance increases at low temperatures, which is opposite to bulk Bi. A TES microcalorimeter with a Bi absorber shows an energy resolution significantly degraded compared to the baseline resolution. A thermal model of the calorimeter based on the present experimental results indicates that a position dependence of the pulse height and rise time degrades the energy resolution. The position dependence is further considered to be due to mismatch between the rise time of pulse which is mostly determined by the thermal conductance between Bi and TES and the thermal diffusion time in the Bi absorber.

Accepted: 2005-04-11, 資料番号: SA1000443000

Fatty acids and related metabolites, comprising several hundreds of molecular species, are an important target in disease metabolomics, as they are involved in various mammalian pathologies and physiologies. Selected reaction monitoring (SRM) analysis, which is capable of monitoring hundreds of compounds in a single run, has been widely used for comprehensive quantification. However, it is difficult to monitor a large number of compounds with different ionization polarity, as polarity switching requires a sub-second period per cycle in classical mass spectrometers. In the present study, we developed and evaluated a comprehensive quantification method for eicosanoids and related compounds by using LC/MS with high-speed continuous ionization polarity switching. The new method employs a fast (30ms/cycle) continuous ionization polarity switching, and differentiates 137 targets either by chromatography or by SRM transition. Polarity switching did not affect the lower limits of quantification, which ranged similarly from 0.5 to 200pg on column. Lipid extracts from mouse tissues were analyzed by this method, and 65 targets were quantitatively detected in the brain, including 6 compounds analyzed in the positive ion mode. We demonstrated that a fast continuous ionization polarity switching enables the quantification of a wide variety of lipid mediator species without compromising the sensitivity and reliability.

Four kinds of 1:1 salts of 3-(4- and 3-alkyl-pyridinium)-1,5-diphenylverdazyl (alkyl = ethyl and methyl) radical cations with the Ni(dmit)2 anion ([p-EtPyDV]+[Ni(dmit)2]− (1), [m-EtPyDV]+[Ni(dmit)2...

There are many serious problems causing a food crisis such as desertification, population explosion, and environmental destruction, suggesting that a severe food crisis will become reality across the globe. Therefore, the transgenic plants, which have tolerance to environmental stresses, may take on greater and greater importance in attempts to increase food production. Aquatic photosynthetic organisms, such as prokaryotic cyanobacteria and eukaryotic green algae, are considered as an evolutionary origin of higher plants and their basic metabolisms including photosynthesis are similar to higher plants. Thereby, stress responsive genes or reactions from these organisms may be exceedingly advantageous sources for creation of stress-tolerant transgenic plants. In this study, the physiological roles and biosynthesis of abscisic acid (ABA), well known as a signal molecule in the adaptation to environmental stresses, in microalgae were investigated from the point of the view of its functional evolution in the response to environmental stresses. Chlamydomonas reinhardtii, a green alga, and Synechocystis sp. PCC 6803, a cyanobacterium, were employed in this study as model organisms. It is expected that this comparative analysis will provide basic information for the creation of stress-tolerant transgenic plants. ABA may act in C. reinhardtii as a signal molecule to induce antioxidant reactions for elimination of reactive oxygen species, but not to induce specific response reactions to mitigate water stresses. In Synechocystis, on the other hand, exogenously added ABA did not influence the growth and gene expression. Moreover, ABA did not relieve growth suppression caused by water and oxidative stresses. From the carotenoid composition and bioinformatic analysis, it can be suggested that the ABA biosynthetic pathway generally found in higher plants exists completely in C. reinhardtii, but incompletely in Synechocystis.

Five kinds of (1:1) and (1:3) salts of 3-(4- and 3-alkyl-pyridinium)-1,5-diphenylverdazyl (alkyl = ethyl and methyl) radical cations with Ni(dmit)2 anion ([p-EtPyDV]+[Ni(dmit)2]− (1), [m-EtPyDV]+[Ni(dmit)2]− (2), [p-MePyDV]+[Ni(dmit)2]− (3), [m-MePyDV]+[Ni(dmit)2]− (4), and [ p - MePyDV ] + [ Ni ( dmit ) 2 ] 3 - ( 5 ) ) (dmit = 1,3-dithiol-2-thione-4,5-dithiolate) have been prepared, and the magnetic susceptibilities (χM’s) have been measured between 1.8 and 300 K. The χM of salt 1 can be well explained by a four-spin linear tetramer model ([p-EtPyDV]+–[Ni(dmit)2]−–[Ni(dmit)2]−–[p-EtPyDV]+) with an end exchange interaction 2J1/kB = −780 K and a central interaction 2J2/kB = −200 K. The formation of magnetic tetramer in salt 1 has been ascertained by the crystal structure analysis of 1. The χM of salt 2 was explained by the sum of the contributions from: (i) a dimer system (2J/kB = −320 K) due to Ni(dmit)2 anions and (ii) the one-dimensional (1D) Heisenberg ferromagnetic linear-chain system (2J kB = +2.5 K) due to verdazyl cations. The χM of salt 3 is similar to that of salt 1, suggesting a formation of magnetic tetramer in salt 3. The χM of salt 4 shows a broad maximum at Tmax = 100 ± 2 K, and can be explained by a 1D Heisenberg antiferromagnetic alternating-chain model with 2J1/kB = −165 K (alternation parameter α = J2/J1 = 0.6). The conductivity (σ) of salts 1, 2, 3 and 4 at 293 K was σ = 2.3 × 10−5, 1.1 × 10−3, 1.4 × 10−5 and 5.4 × 10−6 S cm−1 with an activation energy of EA = 0.28, 0.52, 0.21 and 0.33 eV, respectively. The (1:3) salt 5 (σ = 1.0 S cm−1, EA = 0.048 eV) showed three to five orders of magnitude higher conductivity than those of the (1:1) salts 1–4 at room temperature. All the (1:1) and (1:3) salts 1–5 are new molecular paramagnetic semiconductors.

In the green alga, Chlamydomonas reinhardtii , externally added abscisic acid (ABA) partly released the growth suppression caused by osmotic stress with sorbitol and salt stress with NaCl, in the same way that paraquat did in a previous study. In cells treated with ABA for 24 h, however, the damage caused by a short-period exposure to these water stresses was not reduced in comparison with non-treated cells. Intracellular levels of reactive oxygen species (ROS) in stress-exposed cells were significantly higher than in non-stress-exposed cells. ABA treatment markedly reduced this ROS generation and enhanced gene expression of the antioxidant enzymes, catalase (CAT) and ascorbate peroxidase (APX), in preference to increasing the activities of these enzymes. These results suggest that ABA might only induce response reactions which reduce the oxidative damage derived from exposure to osmotic and salt stresses in C. reinhardtii , but did not act to induce any specific reactions to reduce the damage caused directly by these water stresses. This mitigation of oxidative damage by ABA, probably by the elimination of ROS, might result in the above-mentioned release of growth suppression.

Salicylic acid is used in chemical peeling procedures. However, they have caused many side effects, even salicylism. To achieve a salicylic acid peeling that would be safer for topical use, we recently developed a new formulation consisting of 30% salicylic acid in polyethylene glycol (PEG) vehicle. In an extension of our previous research, we studied the absorption of 30% salicylic acid labeled with 14C in PEG vehicle applied topically to the intact and damaged skin of male hairless mice. An ointment containing 3 mg salicylic acid in 10 mg vehicle was applied to both groups. In animals with intact skin, 1 h after application the plasma concentration of radioactivity was 1665.1 ng eq/ml, significantly lower than the 21437.6 ng eq/ml observed in mice with damaged skin. Microautoradiograms of intact skin showed that the level of radioactivity in the cornified cell layer was similar at 6 h after application. However, in damaged skin, the overall level of radioactivity showed a decrease by 3 h after application. In the carcasses remaining after the treated intact and damaged skin had been removed, 0.09 and 11.38% of the applied radioactivity remained, respectively. These findings confirm that 30% salicylic acid in PEG vehicle is little absorbed through the intact skin of hairless mice, and suggest that salicylism related to absorption through the skin of quantities of topically applied salicylic acid is not likely to occur in humans with intact skin during chemical peeling with this preparation. This new preparation of 30% salicylic acid in PEG vehicle is believed to be safe for application as a chemical peeling agent.

Hairy root cultures of Vinca minor and Ajuga reptans var. atropurpurea could be cryopreserved when the roots were precultured and encapsulated in 2% (w/v) alginate beads with 0.3 M sucrose and 0.5 M glycerol and dehydrated until the bead weight reached 25% of the initial weight before cooling in liquid nitrogen. Preculture and encapsulation of the roots with abscisic acid was effective in increasing the survival rates. For V. minor root tips moreover a sufficiently high survival rate of more than 70% was attained by eliminating glycerol from the preculture medium and dehydration of beads until 23% of the initial weight was reached instead of 25%.