Chimerism and T-cell receptor repertoire analysis after unrelated cord blood transplantation with a reduced-intensity conditioning regimen following autologous stem cell transplantation for multiple myeloma.

A 65-year-old Japanese male was diagnosed as multiple myeloma with Bence Jones kappa type, clinical stage IIIA. His disease status reached partial remission after chemotherapy. Thereafter, he received tandem transplantation, consisting of high-dose chemotherapy with autologous stem cell transplantation (ASCT), followed by unrelated cord blood transplantation (U-CBT). U-CBT with a reduced-intensity conditioning regimen (RI-CBT) was performed in August 2003. HLA mismatch between the patient and the CBT donor was present at two serological loci (B and DR). A total nucleated CBT cell dose of 2.45 x 10(7)/kg body weight was infused on day 0. Graft-vs.-host disease (GVHD) prophylaxis consisted of cyclosporine A and short-term methotrexate. Neutrophil engraftment (>0.5 x 10(9)/l) was obtained on day 46. He developed positive cytomegalovirus antigenemia, grade II acute GVHD involving skin and liver, varicella-zoster virus infection, septic shock, hemorrhagic cystitis caused by adenovirus and acute hepatitis B virus infection after U-CBT. We retrospectively analyzed T-cell receptor (TCR) repertoire diversity and found that TCR repertoire diversity decreased continuously after U-CBT. Therefore, low-TCR repertoire diversity in this patient appears to be associated with various infections caused by immunodeficiency.

Blockade of MCH1 receptor signalling ameliorates obesity and related hepatic steatosis in ovariectomized mice.

BACKGROUND AND PURPOSE: Melanin-concentrating hormone (MCH) is a cyclic orexigenic neuropeptide predominantly expressed in the lateral hypothalamus. We investigated the roles of MCH1 receptor signalling in ovariectomy (OVX)-induced obesity in female C57BL/6J mice, an animal model of postmenopausal obesity. EXPERIMENTAL APPROACH: The effects of blocking signalling via the MCH1 receptor on OVX-induced obesity was investigated by using Mch1r deficient (KO) mice and chronic treatment with a selective MCH1 receptor antagonist. KEY RESULTS: OVX induced body weight gain and increases in the weight of visceral fat and of liver; these effects were attenuated following OVX in Mch1r KO mice. OVX-induced triglyceride (TG) accumulation and elevated expression of lipogenic genes were significantly ameliorated in the liver of Mch1r KO mice. In agreement with these results, chronic i.c.v. infusion of a selective MCH1 receptor antagonist significantly reduced body weight gain, visceral fat and liver weights in OVX mice, and hepatic TG contents and lipogenic gene expression levels were normalized. CONCLUSION AND IMPLICATIONS: Our results indicate that MCH1 receptor signalling is involved in the development of fatty liver, as well as obesity, in OVX mice, and suggest a therapeutic potential for MCH1 receptor antagonists in the treatment of obesity and fatty liver.

Abnormal expansion of naïve B lymphocytes after unrelated cord blood transplantation--a case report.

A 33-year-old woman underwent unrelated cord blood transplantation (U-CBT) for myelodysplastic syndrome (MDS)-related secondary AML. She showed impressive increases in the number of CD19+ B cells in bone marrow and CD19+27-IgD+ B cells in peripheral blood from about 1 month to 3 months after U-CBT. The serum level of IL-6 temporarily increased after transplantation, and this increase seemed to be correlated with the expansion of CD19+ B cells. Although, compared with BMT, little is known about the kinetics of hematological and immunological reconstitution in U-CBT, there was initial B-cell recovery after CBT as some described. This B cell recovery may be associated with a high number of B-cell precursors present in cord blood (CB). The phenomenon of naïve B lymphocyte expansion that we found might be associated with a high number of B-cell precursors present in CB.

Vibrationally resolved fluorescence from organic molecules near metal surfaces in a scanning tunneling microscope.

Intrinsic molecular fluorescence from porphyrin molecules on Au(100) has been realized by using a nanoscale multimonolayer decoupling approach with nanoprobe excitation in the tunneling regime. The molecular origin of luminescence is established by the observed well-defined vibrationally resolved fluorescence spectra. The molecules fluoresce at low "turn-on" voltages for both bias polarities, suggesting an excitation mechanism via hot electron injection from either tip or substrate. The excited molecules decay radiatively through Franck-Condon pi(*)-pi transitions.

Selective assembly on a surface of supramolecular aggregates with controlled size and shape.

The realization of molecule-based miniature devices with advanced functions requires the development of new and efficient approaches for combining molecular building blocks into desired functional structures, ideally with these structures supported on suitable substrates 1-4. Supramolecular aggregation occurs spontaneously and can lead to controlled structures if selective and directional non-covalent interactions are exploited. But such selective supramolecular assembly has yielded almost exclusively crystals or dissolved structures 5; the self-assembly of absorbed molecules into larger structures 6-8, in contrast, has not yet been directed by controlling selective intermolecular interactions. Here we report the formation of surface-supported supramolecular structures whose size and aggregation pattern are rationally controlled by tuning the non-covalent interactions between individual absorbed molecules. Using low-temperature scanning tunnelling microscopy, we show that substituted porphyrin molecules adsorbed on a gold surface form monomers, trimers, tetramers or extended wire-like structures. We find that each structure corresponds in a predictable fashion to the geometric and chemical nature of the porphyrin substituents that mediate the interactions between individual adsorbed molecules. Our findings suggest that careful placement of functional groups that are able to participate in directed non-covalent interactions will allow the rational design and construction of a wide range of supramolecular architectures absorbed to surfaces.

A typical Y1 receptor regulates feeding behaviors: effects of a potent and selective Y1 antagonist, J-115814.

Neuropeptide Y (NPY) is a potent feeding stimulant. The orexigenic effect of NPY might be caused in part by the action of Y1 receptors. However, the existence of multiple NPY receptors including a possible novel feeding receptor has made it difficult to determine the relative importance of the Y1 receptor in feeding regulation. Herein we certified that the Y1 receptor is a major feeding receptor of NPY by using the potent and selective Y1 antagonist (-)-2-[1-(3-chloro-5-isopropyloxycarbonylaminophenyl)ethylamino]-6-[2-(5-ethyl-4-methyl-1,3-thiazol-2-yl)ethyl]-4-morpholinopyridine (J-115814) and Y1 receptor-deficient (Y1-/-) mice. J-115814 displaced (125)I-peptide YY binding to cell membranes expressing cloned human, rat, and murine Y(1) receptors with K(i) values of 1.4, 1.8, and 1.9 nM, respectively, and inhibited NPY (10 nM)-induced increases in intracellular calcium levels via human Y1 receptors (IC(50) = 6.8 nM). In contrast, J-115814 showed low affinities for human Y2 (K(i) > 10 microM), Y4 (K(i) = 640 nM) and Y5 receptors (K(i) = 6000 nM). Intracerebroventricular (ICV) (10-100 microg) and intravenous (IV) (0.3-30 mg/kg) administration of J-115814 significantly and dose-dependently suppressed feeding induced by ICV NPY (5 microg) in satiated Sprague-Dawley rats. Intraperitoneal (IP) administration of J-115814 (3-30 mg/kg) significantly attenuated spontaneous feeding in db/db and C57BL6 mice. Feeding induced by ICV NPY (5 microg) was unaffected by IP-injected J-115814 (30 mg/kg) in Y1-/- mice and was suppressed in wild-type and Y5-/- mice. These findings clearly suggest that J-115814 inhibits feeding behaviors through the inhibition of the typical Y1 receptor. We conclude that the Y1 receptor plays a key role in regulating food intake.

Antithrombotic activity of NSP-513, a novel selective phosphodiesterase 3 inhibitor, on femoral arterial thrombosis induced by physical stenosis and electrical current: comparison of antithrombotic and hemodynamic effects.

NSP-513, a novel potent and selective phosphodiesterase 3 (PDE 3) inhibitor, and cilostazol, a previously developed PDE 3 inhibitor, were compared with respect to antiplatelet, antithrombotic, and hemodynamic effects. In the in vitro antiplatelet aggregation studies, NSP-513 and cilostazol inhibited collagen-induced canine platelet aggregation with median inhibitory concentration (IC50) values of 0.093 and 3.1 miccroM, respectively, and inhibited adenosine diphosphate (ADP)-induced canine platelet aggregation with IC50 values of 0.15 and 12 microM, respectively. For ADP-induced platelet aggregation, the presence of prostaglandin E1 (PGE1; 3 and 10 nM) further decreased the IC50 values for NSP-513 to 0.11 and 0.032 microM, respectively. In ex vivo antiplatelet aggregation studies, orally administered NSP-513 (0.03-1 mg/kg) and cilostazol (50 mg/kg) inhibited collagen-induced canine platelet aggregation. In an in vivo canine femoral arterial thrombosis model, intraduodenally administered NSP-513 (0.01-0.03 mg/ kg) dose-dependently prevented thrombus formation without any changes in blood pressure, heart rate, or bleeding time. In conscious dogs, NSP-513 at oral doses of > or =0.3 mg/kg produced hemodynamic changes such as decreased blood pressure and increased heart rate and LVdP/dt(max). Thus the minimal hemodynamically effective dose of NSP-513 was 0.3 mg/kg, and the hemodynamic effects of this dose were comparable to those of 50 mg/kg of cilostazol. In conclusion, these data suggest that NSP-513 has in vivo selectivity for antiplatelet and antithrombotic activities over hemodynamic activity, and that the selectivity of NSP-513 is higher than that of cilostazol in dogs.

Role of the Y1 receptor in the regulation of neuropeptide Y-mediated feeding: comparison of wild-type, Y1 receptor-deficient, and Y5 receptor-deficient mice.

Neuropeptide Y (NPY) increases food intake through the action of hypothalamic NPY receptors. At least six subtypes of NPY, peptide YY (PYY), and pancreatic polypeptide (PP) receptors have been identified in mice. Although the involvement of Y1 and Y5 receptors in feeding regulation has been suggested, the relative importance of each of these NPY receptors and the participation of a novel feeding receptor are still unclear. To address this issue, we generated a Y1 receptor-deficient (Y1-/-) and a Y5 receptor-deficient (Y5-/-) mouse line in which we directly compared the orexigenic effects of NPY and its analogs after intracerebroventricular (icv) administration. The icv NPY-induced food intake was remarkably reduced in Y1-/- mice, but was not significantly altered by inactivation of the Y5 receptor. The Y1 receptor therefore plays a dominant role in NPY-induced feeding. Stimulation of feeding by moderately selective Y5 agonists [PYY-(3-36), human PP, and bovine PP] was reduced in Y5-/- mice, although food intake did not decrease to vehicle control levels. These results indicate that the Y5 receptor functions as one of the feeding receptors. In addition, the finding that Y5-preferring agonists still induce food intake in Y5-/- mice suggests a role for another NPY receptor(s), including the possibility of novel NPY receptors. Surprisingly, despite the limited efficacy of PYY-(3-36) and PPs at the Y1 receptor, food consumption induced by these agonists was significantly diminished in Y1-/- mice compared with that in wild-type controls. These observations suggest that the feeding stimulation induced by NPY and its analogs may be directly or indirectly modulated by the action of the Y1 receptor. We conclude that multiple NPY receptors, possibly including the novel feeding receptor, are involved in the feeding response evoked by NPY and its analogs. Among them, the Y1 receptor plays a key role in NPY-induced feeding in mice.

Actin motion on microlithographically functionalized myosin surfaces and tracks.

High-resolution e-beam patterning exposure of the surface of poly[(tert-butyl-methacrylate)-co-(methyl methacrylate)]-a common e-beam and deep-UV resist used in semiconductor microlithography-induced sharp changes in the surface hydrophobicity. These differences in hydrophobicity resulted in the selective attachment of heavy meromyosin to hydrophobic, unexposed surfaces. The movement of the actin filaments on myosin-rich and myosin-poor surfaces was statistically characterized in terms of velocity, acceleration, and angle of movement. The actin filaments have a smooth motion on myosin-rich surfaces and an uneven motion on myosin-poor surfaces. Interestingly, an excess of myosin sites has a slowing, albeit mild effect on the motion of the actin filaments. It was also found that the myosin-rich/myosin-poor boundary has an alignment-enforcement effect, especially for the filaments approaching the border from the myosin-rich side. Based on these results, we discuss the feasibility of building purposefully designed molecular motor arrays and the testing of the hypotheses regarding the functioning of the molecular motors.

Disseminated cryptococcosis associated with adrenal masses and insufficiency.

A case of primary adrenal insufficiency with bilateral adrenal masses and meningitis due to disseminated cryptococcosis in a patient with mild non-insulin-dependent diabetes is presented. The diagnosis was made by fine-needle aspiration biopsy cytology. Although the meningitis responded to antifungal therapy, the bilateral adrenal gland enlargement did not change. Reflecting this, cryptococcal antigen titers became negative in CSF, but fell to 1:8 in serum. Although antifungal therapy continued, cryptococcal antigen titer increased both in CSF and serum for 50 days. Because the adrenal glands were the apparent focus for the persistent fungemia, bilateral adrenalectomy was performed. Antifungal therapy for an additional 15 months was needed to achieve negative serum cryptococcal antigen titers. Although adrenal insufficiency due to disseminated cryptococcosis is rare in healthy hosts, it should be included in differential diagnosis of unilateral and bilateral adrenal masses.

Five jumps per day increase bone mass and breaking force in rats.

The effects of jump training on bone morphological and mechanical properties were investigated in immature bones of female Fischer 344 rats. Five-week-old rats were divided into control or five jump-trained groups comprised of 5-, 10-, 20-, 40-, and 100-jump groups, representing the number of jumps per day. The rats were jump-trained 5 days/week for 8 weeks, and the height of jump was increased to 40 cm progressively. The femur and tibia in the 5-jump group had significantly greater fat-free dry weights per body weight and maximum loads at the fracture tests than those in the control group. The tibia in the 5-jump group also had significantly larger cortical area at the cross-sectional analysis. Although a slight tendency toward increase according to the number of jumps per day was observed, there were few differences in bone morphological and mechanical parameters among the 10-, 20-, and 40-jump groups. The present results indicate that a large number of strains per day is not necessary for bone hypertrophy to develop in rats.

Cross-linking of collagen by singlet oxygen generated with UV-A.

Singlet oxygen (1O2), a highly reactive and toxic intermediate, may play a role in photo-induced aging. We examined singlet oxygen generation from hematoporphyrin (HP) with UV-A, by monitoring the emission at 1,268 nm corresponding to 1O2 --> 3O2. Singlet oxygen was formed HP-dose-dependently in this system. We then investigated the reaction of singlet oxygen generated by UV-A irradiation with collagen, which is related to skin elasticity and softness. Collagen from skin was rapidly and dose-dependently cross-linked by singlet oxygen. The reaction was inhibited by NaN3, a selective quencher of singlet oxygen. In contrast, SOD (superoxide dismutase) and mannitol had no effect. These results suggested that cross-linking of collagen was caused by UV-A-generated singlet oxygen, not by any other reactive oxygen species. Compared with another multisubunit protein, alcohol dehydrogenase, collagen was cross-linked much more efficiently. Further, the finding that semicarbazide inhibited cross-linking of collagen showed that cross-links were formed between photooxidized histidyl residues and amino groups. Singlet oxygen generated by UV-A irradiation may contribute to cross-linking of collagen in the process of skin photoaging.

Determination of disodium cromoglycate in human urine by high-performance liquid chromatography with post-column photoirradiation-fluorescence detection.

For the determination of disodium cromoglycate in urine, a fluorimetric method using HPLC post-column photoirradiation has been developed. The mobile phase consisted of a 35 mmol l-1 phosphate buffer (pH 8)-methanol (7 + 3, %v/v) containing 75 mmol l-1 hydrogen peroxide and 20 mmol l-1 18-crown-6. The 18-crown-6 was used for separation adjustment of the disodium cromoglycate in the urine sample. Photoirradiation was carried out in tubing wound around a germicidal light in a reactor equipped with an air-cooling fan. The fluorescence was monitored with excitation at 325 nm and emission at 448 nm. The calibration graph for disodium cromoglycate was linear over the range 38-2340 ng ml-1 using an injection volume of 100 microliters. The pretreatment of the urine samples consisted of diluting and filtering steps. The mean recovery of disodium cromoglycate from urine was 99.1 +/- 2.4% (n = 6).

Participation of reactive oxygen species in phototoxicity induced by quinolone antibacterial agents.

To elucidate the mechanism of phototoxicity induced as a side effect by some of the new quinolone antibiotics, we studied sparfloxacin (SPFX), lomefloxacin, enoxacin, ofloxacin, and ciprofloxacin. We first examined the photosensitized formation of reactive oxygen species such as singlet oxygen (1O2) and superoxide anion (O2-) mediated by the new quinolones. Although a large number of studies have been reported, there is no direct evidence that these drugs generate reactive oxygen species. We employed a near-infrared emission spectrometer to detect 1O2-specific emission (1268 nm), and the nitroblue tetrazolium reduction method to detect O2-. All the quinolones investigated in this study were found to produce 1O2. Four drugs, but not SPFX, produced O2-. We also examined photodynamic DNA strand-breaking activity as a possible mechanism to explain the participation of reactive oxygen species in the phototoxicity of the drugs. All the drugs exhibited photodynamic DNA strand-breaking activity. The inhibitory effect of scavengers of reactive oxygen species indicated that the main active species was 1O2. The DNA strand-breaking activity was correlated not with the 1O2-forming ability, but with the affinity of the drugs for DNA. This result may be due to the short lifetime of 1O2. These data suggested that the phototoxicity of the new quinolones was related to DNA damage caused by reactive oxygen species, especially 1O2.

Control of actin moving trajectory by patterned poly(methylmethacrylate) tracks.

Poly(methylmethacrylate) (PMMA), a photoresist polymer, was found to be useful for immobilizing heavy meromyosin (HMM) molecules while retaining their abilities to support the movement of actin filaments. PMMA substrate was spin-coated on a coverslip, and various shapes of PMMA tracks, such as straight lines, concentric circles, and alphabetical letters, were fabricated by UV photolithography. An observation by a Tapping mode atomic force microscope (AFM) shows that the typical circular tracks were 1-2 microns wide and about 200 nm high. In in vitro motility assay, a solution of HMM molecules was applied to immobilize the molecules on the tracks by adsorption, and movement of actin filaments labeled with tetramethylrhodamine-phalloidin were observed in the presence of ATP by using an epifluorescence microscope and an image-intensified CCD camera. Actin filaments were seen to move precisely only on the PMMA tracks, and their traces drew the exact shapes of the tracks. The mean velocity of actin movement on the PMMA was 4.5 mm/s at 25 degrees C, and it was comparable to that on a conventionally used nitrocellulose film.

Singlet oxygen (1 delta g) generation from coproporphyrin in Propionibacterium acnes on irradiation.

Although singlet oxygen has been postulated to be a highly reactive and toxic intermediate, there has been no evidence of considerable generation of singlet oxygen in vivo level except for special cases. In this work, we firstly measured the near-infrared emission spectra corresponding to the O2(1 delta g) --> O2(3 epsilon g-) transition of singlet oxygen of cutaneous Propionibacterium acnes (P. acnes) porphyrin under laser excitation. A comparison of the singlet oxygen production of coproporphyrin, which is produced predominantly from P. acnes, with that of other photosensitizers revealed coproporphyrin to be a highly efficient singlet oxygen generator under ultraviolet light A irradiation on the skin. These results suggest that singlet oxygen can be generated on the skin surface from P. acnes porphyrin under ultraviolet irradiation and induce serious damage to the skin.

Measurement of 1268 nm emission for comparison of singlet oxygen (1 delta g) production efficiency of various dyes.

Singlet oxygen generation from laser-excited photosensitive dyes was measured directly using a sensitive near-infrared emission spectrometer to monitor the O2(1 delta g)-->O2(3 sigma -g) transition at 1268 nm. The emission intensity was proportional to both the laser power and the concentration of the dyes. The singlet oxygen producing ability of the dyes was compared with that of eosin YS as a standard in methanol. The relative efficiencies of singlet oxygen generation were determined for rose bengal, erythrosine B, phloxine B and eosin YS as 2.39, 1.73, 1.38, 1.00, respectively, while uranine showed no emission in this spectral region. Using rose bengal, erythrosine B, phloxine B and eosin YS, the efficiency of singlet oxygen generation correlated with the photobleaching reaction rate of azo-dyes by these dyes, suggesting singlet oxygen to be a species responsible for causing the photobleaching of azo-dyes. The halogen substituent effect on the efficiency of singlet oxygen generation from laser-excited photosensitive dyes was also examined systematically.

Correlation analysis of blood pressure and platelet aggregation/vascular reactivity in SHRSP, WKY, F1 and backcross generations.

1. Hypofunction of stroke-prone spontaneously hypertensive rat (SHRSP) platelets at developmental ages of hypertension is due to the defective protein (p47) phosphorylation which is mediated by protein kinase C. This study was undertaken to examine the genetic association of platelet functions and vascular reactivity with hypertension using male WKY, SHRSP, F1 and backcross generations at 16-18 weeks of age. 2. The distribution of blood pressure was continuous in each generation. 3. Contraction of mesenteric vascular bed with norepinephrine was positively correlated with blood pressure in the five generations (r = 0.77, n = 128). 4. Thrombin-induced platelet aggregation was inversely correlated with blood pressure (r = -0.87, n = 127). 5. The distribution of platelet aggregation and contraction was continuous in each generation, and backcross generations were not likely to have 1:1 segregation. 6. These results suggest the possibility that platelet hypoaggregability and peripheral vascular resistance are due to the pleiotropic effect of hypertensive genes, or that genes controlling these three characters are closely linked each other.

Blood pressure-related changes of endothelium-dependent relaxation in the aorta from SHRSP at developmental ages of hypertension.

1. Shear stress and flow influence endothelial functions to stimulate the release of endothelium-derived relaxing factor (EDRF). However, it is not well defined how hypertension affects endothelial functions. In this study, blood pressure-related changes of endothelium-dependent relaxation (EDR) in the aorta were examined in SHRSP, WKY and F1 hybrid at developmental ages of hypertension, and hypotensively-treated SHRSP. 2. Acetylcholine-induced EDR of aortic rings was significantly enhanced in 8 week old SHRSP compared with that of age-matched WKY. 3. NG-monomethyl L-arginine (L-NMMA) (1 mmol/L), an inhibitor of nitric oxide, greatly reduced the relaxation in both strains. Indomethacin (10(-5) mol/L), a cyclo-oxygenase inhibitor, did not affect the relaxation at this age. 4. EDR was positively correlated (r = 0.81, n = 22) with blood pressure at 9 weeks of age in WKY, SHRSP and F1 hybrid. 5. A five week hypotensive treatment of SHRSP caused a significant reduction in EDR with decreasing blood pressure. 6. It was concluded that the endothelium releases more EDRF in response to increasing blood pressure to regulate vascular tone at developmental ages of hypertension.