Delayed fragmentation of isolated nucleobases induced by MeV ions.

We evaluated the dissociation of isolated gas-phase nucleobase molecules induced by mega electron volt (MeV)-energy ions to gain fundamental insights into the reactions of nucleobases upon fast ion irradiation. We studied five nucleobase molecules-adenine, guanine, cytosine, thymine, and uracil-as gas-phase targets. We compared the fragmentation patterns obtained from carbon ion impacts with those obtained from proton impacts to clarify the effect of heavy ion irradiation. We also compared the results with electron impact and photoionization results. In addition, we identified several delayed fragmentation pathways by analyzing the correlation between fragment pairs generated from singly and doubly charged intermediate ions. To determine the lifetimes of delayed fragmentation from singly charged intermediate ions, we evaluated the detection efficiencies of the microchannel plate detector for the neutral fragment HCN as a function of kinetic energy using a new methodology. As the first demonstration of this method, we estimated the lifetimes of C5H5N5+ generated by 1.2-MeV C+ and 0.5-MeV H+ collisions to be 0.87 ± 0.43 and 0.67 ± 0.09 µs, respectively. These lifetimes were approximately one order of magnitude longer than those of the doubly charged intermediate ion C5H5N52+.

Fast-ion-induced secondary ion emission from submicron droplet surfaces studied using a new coincidence technique with forward-scattered projectiles.

A mass spectrometric study of secondary ions emitted from droplet surfaces by MeV-energy heavy ion impact was performed to investigate fast-ion-induced molecular reaction processes on liquid surfaces. Herein, a new coincidence technique was developed between secondary ions and scattered projectile ions at a small forward angle. The advantages of this technique were demonstrated by measurement of the collision between 4-MeV C3+ and ethanol droplets. Secondary ion emission probabilities were obtained directly from the coincidence data. Notably, this technique enabled positive fragment ions that had not been identified in previous measurements to be observed by suppressing the strong background originating from gas-phase molecules more than 104-fold. H+, H3O+, C2H5 +, and C2H5O+ were found to be produced as major positive fragment ions, in addition to minor fragments H2 +, C2H3 +, and CH2OH+. Production of these ions suggests that competition between rapid hydrogen ion emission from multiply ionized states and intermolecular proton transfer accompanied by fragmentation through protonated ethanol occurs after fast heavy-ion collisions. Clarification of the positive fragment ions also revealed the characteristic features of negative ions. Negative ions were realized to exhibit higher degrees of fragmentation and reactivity compared with positive ions. Furthermore, the energy loss by forward-scattered ions during droplet penetration was used to evaluate the target thickness at a submicron level. Variations in secondary ion yield, mass distribution, and kinetic energies depending on the penetration length were observed below 1 µm. These results highlight the unknown mechanism of these "submicron effects" observed in secondary ion emission processes as a new phenomenon.

Effects of herpes simplex virus vectors encoding poreless TRPV1 or protein phosphatase 1α in a rat cystitis model induced by hydrogen peroxide.

Enhanced afferent excitability is considered to be an important pathophysiological basis of interstitial cystitis/bladder pain syndrome (IC/BPS). In addition, transient receptor potential vanilloid-1 (TRPV1) receptors are known to be involved in afferent sensitization. Animals with hydrogen peroxide (HP)-induced cystitis have been used as a model exhibiting pathologic characteristics of chronic inflammatory condition of the bladder. This study investigated the effect of gene therapy with replication-defective herpes simplex virus (HSV) vectors encoding poreless TRPV1 (PL) or protein phosphatase 1 α (PP1α), a negative regulator of TRPV1, using a HP-induced rat model of cystitis. HSV vectors encoding green fluorescent protein, PL or PP1α were inoculated into the bladder wall of female rats. After 1 week, 1% HP or normal saline was administered into the bladder, and the evaluations were performed 2 weeks after viral inoculation. In HP-induced cystitis rats, gene delivery of PL or PP1α decreased pain behavior as well as a reduction in the intercontraction interval. Also, both treatments reduced nerve growth factor expression in the bladder mucosa, reduced bladder inflammation characterized by infiltration of inflammatory cells and increased bladder weight. Taken together, HSV-mediated gene therapy targeting TRPV1 receptors could be effective for the treatment of IC/BPS.

Cooling dynamics of photoexcited C6(-) and C6H(-).

We report conclusive evidence of an efficient cooling mechanism via the electronic radiative transitions of hot small molecular anions isolated in vacuum. We stored C6(-) and C6H(-) in an ion storage ring and observed laser-induced electron detachment with delays up to several milliseconds. The terminal hydrogen atom caused a drastic change in the decay profiles. The decay of photoexcited C6H(-) is slow and nonexponential, which can be explained by depletion cooling, whereas that for C6(-) occurs extremely fast, on a time scale below 0.1 ms and can only be explained by electronic radiative cooling via low-lying electronic excited states.

Basic studies toward ultrafast soft x-ray photoelectron diffraction; its application to probing local structure in iodobenzene molecules.

Ultrafast x-ray photoelectron diffraction (UXPD) for free molecules has a promising potential to probe the local structures of the molecules in an element-specific fashion. Our UXPD scheme consists of three steps: (1) near-infrared laser (NIR) with ns pulse duration aligns sample molecules, (2) ultra-violet laser with fs pulse duration pumps the aligned molecules, and (3) soft x-ray free-electron laser (SXFEL) with fs pulse duration probes the molecules by measuring x-ray photoelectron diffraction (XPD) profiles. Employing steps of (1) and (3), we have measured I 3d XPD profiles from ground state iodobenzene aligned by the NIR laser with the SXFEL. Then, we have intensively calculated I 3d XPD profiles with density functional theory, taking degrees of alignments of the molecules into account, to extract a distance between C and I atoms in iodobenzene from the experimental I 3d XPD profiles. Although we have failed to determine the distance from the comparison between the experimental and theoretical results, we have succeeded in concluding that the degeneracies of the initial state eliminate the sensitivity on molecular structure in the I 3d XPD profiles. Thus, the observation of fine structures in the XPD profiles could be expected, if a nondegenerate molecular orbital is selected for a probe of UXPD. Finally, we have summarized our criteria to perform UXPD successfully: (1) to use SXFEL, (2) to prepare sample molecules with the degree of alignment higher than 0.8, and (3) to select a photoemission process from a nondegenerate inner-shell orbital of sample molecules.

Three-dimensional readout of flash x-ray images of living sperm in water by atomic-force microscopy.

The imaging of living specimens in water by x-ray microscopy can be greatly enhanced with the use of an intense flash x-ray source and sophisticated technologies for reading x-ray images. A subnanosecond [corrected] x-ray pulse from a laser-produced plasma was used to record the x-ray image of living sea urchin sperm in an x-ray resist. The resist relief was visualized at high resolution by atomic-force microscopy. Internal structure of the sperm head was evident, and the carbon density in a flagellum was estimated from the relief height.

Atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knee: a prospective multicenter clinical trial in Japan.

BACKGROUND: New tissue-engineering technology was developed to create a cartilage-like tissue in a three-dimensional culture using atelocollagen gel. The minimum 2-year followup outcome of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in knees was reported from the single institution. The present multicenter study was conducted to determine clinical and arthroscopic outcomes in patients who underwent atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knees. METHODS: At six medical institutes in Japan, we prospectively evaluated the clinical and arthroscopic outcomes of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in 27 patients (27 knees) with cartilage lesions on a femoral condyle or on a patellar facet over 24 months. RESULTS: The Lysholm score significantly increased from 60.0 +/- 13.7 points to 89.8 +/- 9.5 points (P = 0.001). Concerning the ICRS grade for arthroscopic appearance, 6 knees (24%) were assessed as grade I (normal) and 17 knees (68%) as grade II (nearly normal). There were few adverse features, except for detachment of the graft in two cases. CONCLUSIONS: We concluded that transplanting chondrocytes in a newly formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee.

A cryogenic linear ion trap beamline for providing keV ion bunches.

A new cryogenic linear ion trap beamline has been constructed and commissioned, which serves to inject cold molecular and cluster ions into the RIKEN cryogenic electrostatic ring (RICE). Ions are created with an electrospray ion source, and a quadrupole mass filter is used for mass-selection prior to trap injection. The radio frequency octupole ion trap can be continuously loaded with ions and features a fast ion extraction mode to create short ion bunches with tens of µs duration. We report here on the simulations and development of the ion trap beamline and validate performance with the moderately heavy molecular cation methylene blue. Characterization of the novel trap design with additional wedge-shaped electrodes was carried out, which includes the determination of the temporal and spatial shape of the ion bunch and the total number of ions after extraction. Finally, these ion bunches are synchronized with the switching of a pulsed high-voltage acceleration device downstream of the trap, where the ions obtain a kinetic energy of up to 20 keV. The preparation and control of the keV ion beam are demonstrated for the ion injection into RICE.

Chitosan-based hyaluronan hybrid polymer fibre scaffold for ligament and tendon tissue engineering.

To establish medical use of tissue engineering technology for ligament and tendon injuries, a scaffold was developed which has sufficient ability for cell growth, cell differentiation, and mechanical properties. The scaffold made from chitosan and 0.1 per cent hyaluronic acid has adequate biodegradability and biocompatibility. An animal experiment showed that the scaffold has less toxicity and less inflammation induction. Furthermore, in-vivo animal experiments showed that the mechanical properties of the engineered ligament or tendon had the possibility to stabilize the joint. It was shown that newly developed hybrid-polymer fibre scaffold has feasibility for joint tissue engineering.

Inverted V-shaped high tibial osteotomy compared with closing-wedge high tibial osteotomy for osteoarthritis of the knee. Ten-year follow-up result.

We compared the results ten years after an inverted V-shaped high tibial osteotomy with those of a historical series of conventional closing-wedge osteotomies. The closing-wedge series consisted of 56 knees in 51 patients with a mean follow-up of 11 years (10 to 15). The inverted V-shaped osteotomy was evaluated in 48 knees in 43 patients at a mean follow-up of 14 years (10 to 19). All the patients were scored using the Japanese Orthopaedic Association rating scale for osteoarthritis of the knee and radiological assessment. The pre-operative grade of osteoarthritis was similar in both groups. Post-operatively, the knee function score was graded as satisfactory in 63% (35) of the closing-wedge group but in 89% (43) of the inverted V-shaped osteotomy group. Post-operative radiological examination showed that delayed union and loss of correction occurred more often after a closing-wedge osteotomy than after an inverted V-shaped procedure. Our study suggests that the inverted V-shaped osteotomy may offer more dependable long-term results than traditional closing-wedge osteotomy.

Fish oil diet affects on oxidative senescence of red blood cells linked to degeneration of spleen cells in mice.

The effect of dietary polyunsaturated fatty acids and alpha-tocopherol supplementation on erythrocyte lipid peroxidation and immunocompetent cells in mice was studied comparatively using seven dietary oils (15% oil/diet, w/w) including fish oil rich in eicosapentaenoic acid (EPA, 20:5, n-3) and docosahexaenoic acid (DHA, 22:6, n-3). A 43% increase in spleen weight, about twice as many spleen cells and no change in the subpopulations of spleen cells, as well as a significant depression of mitogen-induced blastogenesis of both T and B cells in the spleen were observed in mice fed fish oil for 30 days in comparison with soybean oil diet-fed mice. In the fish oil diet-fed mice, membranous lipid hydroperoxide (hydroperoxides of phosphatidylcholine and phosphatidylethanolamine) accumulation as a marker of oxidative senescence in red blood cells (RBC) was 2.7-3.5 times higher than that in mice fed soybean oil, although there was no difference in the plasma phosphatidylcholine hydroperoxide concentration. In spite of the supplementation of alpha-tocopherol to up to 10 times the level in the basal diet, the degeneration of spleen cells and the stimulated oxidative senescence of RBC found by the fish oil feeding could not be prevented. The results suggest that oral intake of excess polyunsaturated fatty acids, i.e. EPA and DHA, in a fish oil diet can lead to acceleration of membrane lipid peroxidation resulting in RBC senescence linked to the lowering of immune response of spleen cells, and that supplementation of alpha-tocopherol as antioxidant does not always effectively prevent such oxidative degeneration as observed in spleen cells and RBC in vivo.

The effect of dietary autoxidized oils on immunocompetent cells in mice.

Effects of dietary autoxidized oil on immunocompetent cells, such as splenocytes and thymocytes, were studied in mice. When the autoxidized methyl linoleate was administered orally to male C57BL/6 mice in a single dose, the DNA synthesis of thymocytes was remarkably depressed 1 day after the treatment, and then the mitogenic response to concanavalin A of splenocytes was increased 3 days after the dose. With long-term (90 days) feeding of slightly autoxidized soybean oil (with a peroxide value of 150 mequiv/kg) in mice, the DNA synthesis of thymocytes was depressed and the mitogenic response to concanavalin A of splenocytes was increased. No effect was observed on plasma glutamic acid-oxaloacetic acid transaminase and glutamic acid-pyruvic acid transaminase levels, nor on liver thiobarbituric acid reactants due to the dose of autoxidized soybean oil. These findings indicate that oral intake of autoxidized oil affects immunocompetent cells and causes depression of the DNA synthesis of thymocytes in mice.

Membrane oxidative metabolism of human eosinophilic cell line EoL-1 in response to phorbol diester and formyl peptide: synergistic augmentation by interferon-gamma and tumor necrosis factor.

Human eosinophilic cell line EoL-1 was studied using luminol-dependent chemiluminescence (CL) for the ability to produce a respiratory burst upon stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). TPA, a potent activator of protein kinase C, induced a biphasic CL response in EoL-1. Treatment of EoL-1 with interferon-gamma (IFN-gamma) for 5 days dramatically enhanced TPA-inducible CL, and IFN-alpha A had a similar effect. Neither IFN-gamma nor IFN-alpha A strongly inhibited EoL-1 cell growth. Tumor necrosis factor (TNF) also enhanced TPA-inducible CL response of EoL-1 and, furthermore, was quite inhibitory to EoL-1 cell growth. The effects of IFN-gamma and TNF were synergistic, whereas those of IFN-alpha A and TNF were additive. Superoxide dismutase completely abrogated TPA-induced CL, but sodium azide suppressed only the late phase of CL. EoL-1 pretreated with IFN-gamma, IFN-alpha A, or TNF also became capable of producing CL response to a chemotactic peptide (fMLP). The effects of IFN-gamma and TNF were again synergistic. EoL-1 cells treated with IFN-gamma, IFN-alpha A, or TNF had abundant cytoplasm, but only TNF increased cells having distinct eosinophilic granules. IFN-gamma but not IFN-alpha A enhanced the cytological effect of TNF. It was further demonstrated that treatment of EoL-1 with IFN-gamma and TNF strongly increased the binding sites for phorbol diesters and also dramatically induced the surface expression of fMLP receptors. IFN-gamma had, however, little effect on the number or the ligand-binding affinity of TNF receptors on EoL-1. Thus, EoL-1 may provide a useful experimental model for the study of differentiation and regulation of human eosinophils.

Mutations of K-ras but not p53 genes in biliary duct and pancreatic duct carcinomas induced in hamsters by cholecystoduodenostomy with dissection of the common duct followed by N-nitrosobis(2-oxopropyl)amine.

An experimental model for the induction of extrahepatic biliary duct carcinomas in hamsters given cholecystoduodenostomy with dissection of the extrahepatic duct at the distal end of the common duct (CDDB) followed by N-nitrosobis(2-oxopropyl)amine (BOP) has been reported [Tajima et al. (1994) Jpn. J. Cancer Res., 85, 780-788]. The CDDB procedure greatly accelerates cell turnover in the biliary epithelium. In the present experiment, mutations of K-ras and p53 genes in the induced lesions were investigated by the reverse transcriptase-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) method followed by direct sequencing. Mutations of K-ras, involving a G to A transition in second position of codon 12 of K-ras exon 1, were detected in six out of eight (75%) extrahepatic bile duct carcinomas and six out of eleven (54.5%) pancreatic duct carcinomas. However, no mutations of p53 were observed in either tumor type. The results indicate an association between anomalous pancreaticobiliary junction and development of biliary carcinomas that may be pertinent to the human situation, and indicate that conditions of the model predispose to mutations occurring in K-ras but not p53.

Infrequent Ki-ras and an absence of p53 mutations in hepatocellular carcinomas induced by a choline deficient L-amino acid defined diet in rats.

Mutations of Ki-ras and p53 genes in hepatocellular carcinomas (HCCs) induced by the choline deficient L-amino acid defined (CDAA) diet in rats were investigated by polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) analysis followed by direct sequencing. Male Fischer 344 rats, 6 weeks old, were continuously given a CDAA diet for 70 weeks and then sacrificed. Macroscopically detectable nodules which were histologically confirmed to be well-differentiated HCCs were dissected free from the surrounding tissue and subjected to gene mutation analysis along with samples of non-tumor areas. Conformational change in the Ki-ras gene was detected in 1 out of 7 HCCs, involving a GGC to GTC transversion at codon 13. No p53 mutations were detected in 7 HCCs and also neither Ki-ras nor p53 mutations were found in non-tumor areas. The results suggest that neither Ki-ras nor p53 genes play an important role in hepatocarcinogenesis caused by long term expose to a CDAA diet in rats.

Increased telomerase activity in intrahepatic cholangiocellular carcinomas induced by N-nitrosobis(2-oxopropyl)amine in hamsters.

Telomerase activities in intrahepatic cholangiocarcinomas induced by N-nitrosobis(2-oxopropyl)amine (BOP) in female hamsters were determined using a telomeric repeat amplification protocol (TRAP) assay followed by densitometric quantification. Each determination was repeated to confirm the results and telomerase activity was also detected by gel electrophoresis. An increase was evident in all of 10 cholangiocarcinomas examined, with levels ranging from 2.48 to 4.40 times the normal liver value by densitometric quantification. This finding of a consistent increase suggests that telomerase activation is involved in the development of intrahepatic cholangiocarcinomas and immortalization of cancer cells.

Forskolin-stimulated adenylylcyclase activity: a marker to assess islet cell viability following cold storage in different solutions and to predict islet cell function following transplantation.

For clinical islet cell transplantation, short-term storage of islet cells is likely to be necessary, and it is imperative that the islet cells be kept as viable as possible during the period. However, there are little data on which preservative solutions are most suitable for the storage of islet cells after isolations or before transplantation. To estimate islet cell viability and transplantation success rate in the present study, adenylylcyclase activity was measured with a rapid new fluorometric assay in rat islet cells prior to transplantation, because cAMP plays an essential role in determining islet beta-cell viability and responsiveness to various hormonal stimuli. Adenylylcyclase activity was measured in islet cells stored for different periods of time 0, 3, 16, 24, 48, 96 h) and in different preservative solutions. Approximately 1,000 islet cells from each preservation group using University of Wisconsin (UW) solution were transplanted to streptozotocin-induced diabetes mellitus (DM) rats. Transplant success was evaluated by measuring blood glucose levels. Preoperative adenylylcyclase activity was compared with posttransplant islet cell function. The adenylylcyclase activity of UW solution was significantly higher than that of Euro-Collins solution and lactate-Ringer's solution through the different preservation time periods. Preoperative adenylylcyclase activity correlated well with posttransplant islet cell function in a rat model of DM. We conclude that adenylylcyclase activity can be used as a marker to assess islet cell viability as well as differences in preservation media and may predict islet cell transplant success.

Inhibitory effects of beta-carotene, palm carotene, and green tea polyphenols on pancreatic carcinogenesis initiated by N-nitorsobis(2-oxopropyl)amine in Syrian golden hamsters.

The effects of alpha-carotene, beta-carotene, palm carotene, and green tea polyphenols (GTP) on the progression stage of pancreatic carcinogenesis after rapid production of ductal lesions were studied in Syrian hamsters. Dose threshold inhibitory effects were noted for beta-carotene, 25 ppm, and palm carotene, 40 ppm, which includes 24 ppm beta-carotene reducing the numbers of putative preneoplastic lesions of duct epithelial hyperplasia and atypical hyperplasia, as well as carcinoma in situ and invasive carcinomas. GTP at doses of 500 and 5000 ppm, but not 100 ppm, also significantly decreased the numbers of hyperplasia and total duct lesions. Combined administration of 40 ppm palm carotene, and 50 ppm GTP similarly inhibited the lesion development. Alpha-carotene, however, did not affect pancreatic carcinogenesis. The results suggest that chemopreventive effects are exerted by beta-carotene and GTP above critical doses and that combined administration of palm carotene and GTP might be a candidate chemoprevention strategy for pancreatic cancer in humans.

Compressive compared with tensile loading of medial collateral ligament scar in vitro uniquely influences mRNA levels for aggrecan, collagen type II, and collagenase.

To test the hypothesis that loading conditions can be used to engineer early ligament scar behaviors, we used an in vitro system to examine the effect that cyclic hydrostatic compression and cyclic tension applied to 6-week rabbit medial collateral ligament scars had on mRNA levels for matrix molecules, collagenase, and the proto-oncogenes c-fos and c-jun. Our specific hypothesis was that tensile stress would promote more normal mRNA expression in ligament whereas compression would lead to higher levels of mRNA for cartilage-like molecules. Femur (injured medial collateral ligament)-tibia complexes were subjected to a hydrostatic pressure of 1 MPa or a tensile stress of 1 MPa of 0.5 Hz for 1 minute followed by 14 minutes of rest. On the basis of a preliminary optimization experiment, this 15-minute testing cycle was repeated for 4 hours. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed for mechanically treated medial collateral ligament scars with use of rabbit specific primer sets for types I, II, and III collagen, decorin, biglycan, fibromodulin, versican, aggrecan, collagenase, c-fos, c-jun, and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. Cyclic hydrostatic compression resulted in a statistically significant increase in mRNA levels of type-II collagen (171% of nonloaded values) and aggrecan (313% of nonloaded values) but statistically significant decreases in collagenase mRNA levels (35% of nonloaded values). Cyclic tension also resulted in a statistically significant decrease in collagenase mRNA levels (66% of nonloaded values) and an increase in aggrecan mRNA levels (458% of nonloaded values) but no significant change in the mRNA levels for the other molecules. The results show that it is possible to alter mRNA levels for a subset of genes in scar tissue by supplying unique mechanical stimuli in vitro and thus that further investigation of scar engineering for potential reimplantation appears feasible.

Biomechanical effects of stress shielding of the rabbit patellar tendon depend on the degree of stress reduction.

A rabbit model was used to discover whether the effects of stress shielding on the mechanical properties of the patellar tendon depend on the degree of stress reduction. Ninety mature female Japanese White rabbits were divided into three groups: completely stress-shielded partially stress-shielded and sham-operation and contralateral controls. In the experimental groups, tension applied to the patellar tendon was 0%, approximately 30%, and 100% of the normal tension, respectively, with a polyester artificial ligament. Tensile tests were carried out on patella-patellar tendon-tibia complexes harvested 1, 2, 3, 6, or 12 weeks after surgery. The tensile strength decreased in comparison with the sham-operation group to 50.2, 13.5, 9.7, and 20.7% in the completely stress-shielded group and to 75.2, 57.6, 59.6, 57.3, and 72.9% in the partially stress-shielded group. The tensile strength in the completely stress-shielded group was significantly less than that in the partially stress-shielded group at 1, 2, 3, and 6 weeks. The cross-sectional area of the patellar tendon significantly increased to 132, 206, 237, and 136% in the completely stress-shielded group and to 136, 170, 175, 155, and 127% in the partially stress-shielded group compared with the sham-operation group. The cross-sectional area of the completely stress-shielded tendon was significantly larger than that of the partially stress-shielded tendon at 1, 2, and 3 weeks. This study demonstrated that effects of stress shielding on the mechanical properties of the patellar tendon were dependent on the degree of stress shielding.