Higher NORs-expression in lymphocyte of trisomy 21 babies/children: in vivo evaluation

Extra chromosome 21 of Down syndrome (DS) or trisomy 21 patients contains an average of 40 extra copies of rRNA genes and the in vivo regulation of these genes is not known. The objective of this work is to compare the NORs expression in interphase nuclei in non-stimulated lymphocytes of DS patients and healthy controls. Because the AgNOR staining is the indicator of the active rRNA genes, comparison of the image analysis values of AgNORs area between DS's and healthy controls' interphase lymphocytes is considered to be sufficient to evaluate the level of rDNA activities in the two groups. The Nucleolus Organizer Regions area/Total Nuclear area (NORa/TNa) was calculated using a computer program designed by us. 100 consecutive NORa/TNa per individual were evaluated. We report that 24 DS children's peripheral lymphocytes show significantly higher NORa/TNa mean value (6.32 +/- 1.77%) than that of the 20 healthy controls' cells (5.31 +/- 1.34%) (2-tailed Mann-Whitney U test, z = 19.4, P = 0.000). The same is true for the nucleolus (AgNOR spot) number per nucleus. The mean value of nucleoli number per nucleus in DS lymphocytes was significantly higher than that of the controls: z = 14.6, P = 0.000. In conclusion, extra rRNA genes on the chromosome 21 are not down-regulated in DS patients' lymphocytes. Rather, extra NORs expressions in 'in vivo' condition contribute to the increase of AgNORs area and AgNOR spots number per nucleus. This is the first work on the comparison of NORs activities in resting (non-stimulated) interphase lymphocytes between DS and healthy controls.     

AgNOR increase in buccal epithelial cells of trisomy 21 infants

The aim of this study is to compare the Argilophilic Nucleolus Organizer Regions (AgNORs) level between Down syndrome (DS) patients and controls in a tissue sharing the same embryonic origin with the central nervous system and compare the results with those obtained recently by us from DS's lymphocytes. For this, buccal desquamating epithelial cells well known as the ectodermic origin were used. Since the AgNOR staining intensity is an indicator of the ribosomes biosynthesis rate, comparison of the image analysis values of the AgNOR area/total nuclear area (NORa/TNa) in buccal desquamating epithelial cells of DS patients and controls provided a plausible conclusion about the regulation/deregulation of the rRNA genes (rDNA) in these cells of DS babies/infants. The (NORa/TNa) proportion was calculated using an in-house computer program. Fifty buccal desquamating cells were analysed for each individual to determine the average NORa/TNa value per individual. In contrast to healthy controls, NORa/TNa proportion value of buccal epithelial cells from DS patients found significantly higher than that of the controls: (4.08 ± 1.16)% and (2.13 ± 0.55)%, respectively. This 92% increase is far higher than the expected value due to the extra rRNA genes on the extra-chromosome 21. Finally DS babies/infants exhibit very higher AgNOR expression increase in their buccal epithelial cells compared to controls. This is the first study that is available on the comparison of AgNOR expression levels in buccal epithelial cells between DS infants and their controls.     

Study of the nucleolar cycle and ribosomal RNA distribution during meiosis in triatomines (Triatominae, Heteroptera)

Aspects of nucleolar activity during spermatogenesis were assessed in three triatomine species (Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans) using cytochemical and fluorescent staining techniques. Toluidine blue and a variant of critical electrolytic concentration (CEC) allowed the discrimination of rRNA providing structural details of the nucleolus and RNA distribution during meiotic cell division. Acridine orange fluorochrome staining permitted the differentiation of nucleic acids, and silver-ion impregnation made possible the observation of pre-nucleolar bodies (PNBs). Our results support the phenomenon known as "persistence of the nucleolar material", and the hypothesis of post-meiotic reactivation of rRNA genes. Nucleolar organizer regions (NORs) were observed in some metaphasic spermatogonial chromosomes in P. megistus and T. infestans. In P. megistus at diplotene-diakinesis, NORs were also detected in one of the sex chromosomes and in an autosome. Therefore, it may be inferred that, in triatomines, the nucleolus does not completely disappear, but persists in the form of small bodies that get together to form the next nucleolar cycle which, in the case of meiosis, will be completed if fertilization occurs and a new zygote is formed.     

IRRADIATION OF HUMAN LYMPHOCYTES WITH He-Ne LASER INCREASES THE BLAST Ⅰ TRANSFORMATION CAUSED BY PHYTOHEMAGGLUTSNIN

The irradiation of human lymphocytes with a He-Ne laser(632.8nm,56J/m~2) elevated the DNA synthesis levol in phytohemagglutinin-treated cells,but didnot itself cause the blasttransformation.The boosting effect of irradition was especiallypronounced in case of suboptimal PHA concentrations(0.5and 1μg/ml).     

Improved measurement of the form factors in the decay lambda+c-->lambda + nue

Using the CLEO detector at the Cornell Electron Storage Ring, we have studied the distribution of kinematic variables in the decay lambda(+)(c)lambda--> e(+)nu(e). By performing a four-dimensional maximum likelihood fit, we determine the form factor ratio, R= f(2)/f(1) = -0.31 +/- 0.05(stat) +/- 0.04(syst), the pole mass, M(pole) = [2.21 +/- 0.08(stat) +/- 0.14(syst)] GeV/c(2), and the decay asymmetry parameter of the lambda(+)(c), alpha (lambda(c)) = -0.86 +/-0.03(stat) +/- 0.02(syst), for q(2) = 0.67 (GeV/c(2))(2). We compare the angular distributions of the lambda(+)(c) and lambda(-)(c) and find no evidence for CP violation: A(lambda(c)) = (alpha(lambda(c)) + alpha (lambda(c)))/(alpha(lambda(c))-alpha(lambda(c))) = 0.00 +/- 0.03(stat) +/- 0.01(syst) +/- 0.02, where the third error is from the uncertainty in the world average of the CP-violating parameter, A(lambda), for ppi(-).     

Software compensation of eddy current fields in multislice high order dynamic shimming

Dynamic B(0) shimming (DS) can produce better field homogeneity than static global shimming by dynamically updating slicewise shim values in a multislice acquisition. The performance of DS however is limited by eddy current fields produced by the switching of 2nd and 3rd order unshielded shims. In this work, we present a novel method of eddy field compensation (EFC) applied to higher order shim induced eddy current fields in multislice DS. This method does not require shim shielding, extra hardware for eddy current compensation or subject specific prescanning. The interactions between shim harmonics are modeled assuming steady state of the medium and long time constant, cross and self term eddy fields in a DS experiment and 'correction factors' characterizing the entire set of shim interactions are derived. The correction factors for a given time between shim switches are shown to be invariable with object scanned, shim switching pattern and actual shim values, allowing for their generalized prospective use. Phantom and human head, 2nd and 3rd order DS experiments performed without any hardware eddy current compensation using the technique show large reductions in field gradients and offsets leading to significant improvements in image quality. This method holds promise as an alternative to expensive hardware based eddy current compensation required in 2nd and 3rd order DS.     

Freeze-drying of human platelets: influence of saccharide, freezing rate and cell concentration

Freeze-drying of human platelets is one potentially ideal approach for long-term preservation of platelets. In this study, effects of concentration and type of saccharides, freezing rate and initial cell concentration on the recovery of freeze-dried platelets were investigated. Annexin V binding platelet activation assays, scanning electron microscopy and platelet aggregation upon thrombin (1 U/ml) addition were used to evaluate the effectiveness of platelet freeze-drying. The numerical recovery of freeze-dried platelets was reached as high as 93.0+/-5.2 percent and the recovery of nonactive platelets was reached up to 85.7 +/- 3.4 percent in the presence of 1% BSA and 20% trehalose. Frozen by shelf pre-cooling was the best way to freeze the sample in this study and the numerical recovery of freeze-dried platelets was reached 93.0 +/- 5.2 percent at about 10 degree C/min. When the platelet concentration was increased from 0.2 to 4x10(9) platelets/ml, recovery remained higher than 81.4 percent. The morphology of freeze-dried and rehydrated platelets was intact but a little rounder compared with fresh platelets. The maximum aggregation rate to thrombin (1 U/ml) of freeze-dried platelets was 83.9 percent of the fresh ones, but aggregation speed was 43.0 percent of the fresh ones. Further research on rehydration process and scale up are required.     

Diclofenac sodium-loaded solid lipid nanoparticles prepared by emulsion/solvent evaporation method

The preparation of solid lipid nanoparticles (SLNs) suffers from the drawback of poor incorporation of water-soluble drugs. The aim of this study was therefore to assess various formulation and process parameters to enhance the incorporation of a water-soluble drug (diclofenac sodium, DS) into SLNs prepared by the emulsion/solvent evaporation method. Results showed that the entrapment efficiency (EE) of DS was increased to approximately 100% by lowering the pH of dispersed phase. The EE of DS-loaded SLNs (DS-SLNs) had been improved by the existence of cosurfactants and increment of PVA concentration. Stabilizers and their combination with PEG 400 in the dispersed phase also resulted in higher EE and drug loading (DL). EE increased and DL decreased as the phospholipid/DS ratio became greater, while the amount of DS had an opposite effect. Ethanol turned out to be the ideal solvent making DS-SLNs. EE and DL of DS-SLNs were not affected by either the stirring speed or the viscosity of aqueous and dispersed phase. According to the investigations, drug solubility in dispersion medium played the most important role in improving EE.     

Con-A诱发的淋巴细胞化学发光

本文以刀豆素 A(ConA)为诱发剂,研究了绵羊肺淋巴液中的淋巴细胞鲁米诺依赖的化学发光现象.结果表明,ConA诱发的淋巴细胞化学发光测定具有迅速、简便和敏感等优点,可作为淋巴细胞活化的检测方法.淋巴细胞悬液(106～107个细胞/ml)1ml、ConA溶液(2mg/ml)0.1ml、鲁米诺溶液(10-3M)50μl为最佳测定体系.多形核白细胞(PMN)混杂可明显增强发光体系的发光强度.因此,测定外周血中的淋巴细胞化学发光,细胞的纯化是一个不可忽视的问题.消炎痛可抑制其发光,说明发光可能与花生四烯酸代谢有关.     

A modified high-output,size-selective aerosol generator

A compact and commercial aerosol generator capable of generating narrowly size-distributed aerosols with high mass concentrations was designed, fabricated and tested. The aerosol generator, consisting of a Delavan simplex nozzle (Model 30610-4), an L-shaped settling chamber and a virtual impactor with a clean air core, was modified and improved from Chein and Lundgren's work [20] to be more compact and readily commercial. The performance of the aerosol generator was evaluated using corn-oil, sodium chloride and uranine solutions. The results indicated that the cornoil droplets produced by the generator had a mass medium aerodynamic diameter (MMAD) of 7.20 ± 0.32 μm with a geometric standard deviation (GSD) of 1.48 ± 0.01 and the aerosol generation rate was 13.8 ± 1.3 mg/min. Solid aerosols generated from NaCl solution were found to have an MMAD in the range 1.39–4.88 μm with a GSD of 1.34–1.47 with the volumetric solution concentration varying from 0.1% to 9%. At the same time, the aerosol generation rate varied from 0.27 ± 0.05 to 15.8 ± 1.8 mg/min. depending on the solution concentration and the particle size produced. In addition, a 0.01% uraniane solution was tested to generate a submicron aerosol with an MMAD of 0.93 μm and a GSD of 1.48.     

Measurement of the branching fractions of exclusive _B-->D(*)(pi)l-_nu l decays in events with a fully reconstructed B meson

We report a measurement of the branching fractions for _B-->D(*)(pi)l- _nu(l) decays based on 341.1 fb(-1) of data collected at the Upsilon(4S) resonance with the BABAR detector at the SLAC PEP-II e+ e- storage rings. Events are tagged by fully reconstructing one of the B mesons in a hadronic decay mode. We obtain B(B- -->D(0)l-_nu(l)=(2.33+/-0.09(stat)+/-0.09(syst)%, B(B- -->D(*0)l-_nu(l)=(5.83+/-0.15(stat) +/-0.30(syst) %, B(_B(0)-->D+l-_nu(l)=(2.21+/-0.11(stat) +/-0.12(syst)%, B(_B(0)-->D(*)l-_nu(l)=(5.49+/-0.16(stat)+/-0.25(syst)%, B(B- -->D+pi-l-_nu(l)=(0.42+/-0.06(stat)+/-0.03(syst)%, B(B- -->D(*)+pi-l-_nu(l)=(0.59+/-0.05(stat)+/-0.04(syst)%, B(_B(0)-->D(0)pi+l-_nu(l)=(0.43+/-0.08(stat)+/-0.03(syst)%, and B(_B(0)-->D(*0)pi+l-_nu(l)=(0.48+/-0.08(stat)+/-0.04(syst)%.     

Cryopreservation of seeds of a Thai orchid (Doritis pulcherrima lindl. ) by vitrification

Seeds from selfing of a Thai orchid (Doritis pulcherrima Lindl.) were successfully cryopreserved in liquid nitrogen (LN) using the vitrification method. Seeds from 3-month-old pods were sufficiently dehydrated in 2 ml cryotubes filled with highly concentrated vitrification solution (PVS2) at 25 +/- 2 degree C for 50 min. The seeds were then rapidly plunged into LN. After rapid warming, the PVS2 solution was replaced with 0.5 ml of 1.2M sucrose in modified Vacin and Went (1949) (VW) solution and kept at 25 +/- 2 degree C for 20 min prior to transfer on VW agar medium. About 62% of cryopreserved seeds treated with PVS2 solution were able to develop into normal seedlings while without that treatment there was no survival. This vitrification protocol appears to be a promising technique for the cryopreservation of some Thai orchid germplasm     

Observation of the radiative decay D0-->phigamma

We report the observation of the decay D0-->phigamma with a statistical significance of 5.4sigma in 78.1 fb(-1) of data collected by the Belle experiment at the KEKB e+e- collider. This is the first observation of a flavor-changing radiative decay of a charmed meson. The Cabibbo- and color-suppressed decays D0-->phipi(0), phieta are also observed for the first time. We measure branching fractions B(D0-->phigamma)=[2.60(+0.70)(-0.61)(stat)+0.15-0.17(syst)] x 10(-5), B(D0-->phipi(0))=[8.01+/-0.26(stat)+/-0.47(syst)] x 10(-4), and B(D0-->phieta)=[1.48+/-0.47(stat)+/-0.09(syst)] x 10(-4).     

MR imaging of the her2/neu and 9.2.27 tumor antigens using immunospecific contrast agents

Molecular imaging of tumor antigens using immunospecific magnetic resonance (MR) contrast agents is a rapidly evolving field, which can potentially aid in early disease detection, monitoring of treatment efficacy, and drug development. In this study, we designed, synthetized, and tested in vitro two novel monocrystalline iron oxide nanoparticles (MION) conjugated to antibodies against the her2/neu tyrosine kinase receptor and the 9.2.27 proteoglycane sulfate. MION was synthetized by coprecipitation of iron II and iron III salts in 12-kD dextran solution; antibody coupling was performed by reductive amination. The relaxivity of the conjugates was 24.1-29.1 mM(-1) s(-1), with 1.8 to 2.1 antibody molecules per nanoparticle. A panel of cultured melanoma and mammary cell lines was used for testing. The cells were incubated with the particles at 16-32 microg Fe/ml in culture medium for 3 h at 37 degrees C, and investigated with immune fluorescence, transmission electron microscopy (TEM), MRI of cell suspensions in gelatine, and spectrophotometric iron determination. All receptor-positive cell lines, but not the controls, showed receptor-specific immune fluorescence, and strong changes in T(2) signal intensity at 1.5 T. The changes in 1/T(2) were between 1.5 and 4.6 s(-1) and correlated with the amount of cell-bound iron (R = 0.92). The relaxivity of cell-bound MION increased to 55.9 +/- 10.4 mM(-1) s(-1). TEM showed anti-9.2.27 conjugates binding to the plasma membrane, while the anti-her2/neu conjugates underwent receptor-mediated endocytosis. In conclusion, we obtained receptor-specific T(2) MR contrast with novel covalently bound, multivalent MION conjugates with anti-9.2.27 and anti-her2/neu to image tumor surface antigens. This concept can potentially be expanded to a large number of targets and to in vivo applications.     

Ammonium nitrate in the culture medium influences regeneration potential of cryopreserved shoot tips of Holostemma annulare

Influence of NH4NO3 in the pre-freeze and post-freeze culture medium and 2 or 30 day preconditioning in the presence of 0.5 M sucrose on regeneration of shoot tips of Holostemma annulare following cryopreservation using an encapsulation-dehydration protocol was studied. A long preconditioning phase of 30 days significantly reduced tissue water and improved post-freeze recovery of shoot tips. Under the long preconditioning treatment, Murashige & Skoog (MS) medium free of NH4NO3 (MS-3) allowed maximum regeneration (59%) of liquid nitrogen (LN) exposed shoot tips with less frequency of callusing (10.4%) after 45 days of post-freeze culture. Corresponding desiccated control shoot tips showed 85-90% regeneration. A 3.75 mM NH4NO3 concentration (MS-4) favoured 72-89% and 43-47% regeneration after desiccation and LN exposure respectively. The standard MS medium with 20.6 mM NH4NO3 (MS-1) allowed poor regeneration after desiccation (39-53%) as well as LN exposure (8-23%). The study reveals the importance of reducing ammonium nitrate in the culture medium to get maximum recovery of cryopreserved shoot tips of Holostemma annulare.     

Flow injection determination of metoclopramide based on KMnO4—HCHO chemiluminescence in a micellar medium

A novel flow injection chemiluminescence (CL) analysis method for the determination of metoclopramide in the presence of sodium dodecyl sulfonate (SDS) surfactant micelles is described. This method is based on the luminescent properties of the KMnO₄-HCHO-metoclopramide in acid medium sensitized by SDS. The optimized experimental conditions were evaluated and the possible mechanism was discussed. The CL increase is linearly related to the concentration of metoclopramide in the range 0-80.0 μg/ml with a detection limit of 31.3 ng/mL (S/N=3).The relative standard deviation for 20.0 μg/ml samples was 2.6% (n=11). The proposed method has been successfully applied to the determination of metoclopramide in tablets.     

In vitro labeling and MRI of mesenchymal stem cells from human umbilical cord blood

OBJECTIVE: The aim of this study was to label human umbilical cord blood mesenchymal stem cells (MSCs) with poly-l-lysine (PLL)-conjugated superparamagnetic iron oxide particles and to obtain magnetic resonance (MR) images of the labeled MSCs' suspension at 1.5 T. MATERIAL AND METHODS: PLL was conjugated with iron oxide to form superparamagnetic particles called Fe(2)O(3)-PLL. Human umbilical cord blood MSCs were isolated, purified, expanded and incubated with Fe(2)O(3)-PLL. Prussian blue stain was performed to show intracellular iron; spectrometry was used to quantify iron uptake within cells. Tetrazolium salt (MTT) assay was applied to evaluate toxicity and proliferation of MSCs labeled with various concentrations of Fe(2)O(3)-PLL. The cell apoptosis rate was determined by annexin V/propichium iodide (PI) double staining method. Vials containing cells underwent MR imaging (MRI) with T(1), T(2) and T(2)* weighted MRI. RESULTS: Iron-containing intracytoplasmatic vesicles could be observed clearly with Prussian blue staining in all samples except the unlabeled control. The iron content per cell determined by spectrometry was 64.51+/-10.32 pg. Among MSCs with and without labeling of various concentrations of Fe(2)O(3)-PLL, MTT values of light absorption had no statistically significant difference (Kruskal-Wallis test, chi(2)=10.35, P=.17). A concentration at 20 mug/ml of iron appeared most suitable for incubating cells. Of labeled and unlabeled MSCs, the early [annexin V-fluorescein isothiocyanate (FITC)-positive/PI-negative] and late (annexin V-FITC-positive/PI-positive) apoptotic cells were 10.34+/-0.43%/11.36+/-1.30% and 4.01+/-1.76%/2.98+/-1.37%, respectively, and there were no significant differences between them (P>.05). T(2) weighted image (WI) and T(2)*WI demonstrated significant decrease of signal intensity (SI) in vials containing 1 x 10(6) (1 day), 1x10(6) (8 days) and 5 x 10(5) labeled cells, in comparison with unlabeled cells (P<.05). The percentage change of SI (DeltaSI) was significantly higher in 10(6) labeled cells after 1-day culture than that in the same number of labeled cells after 8-day culture and that in 5 x 10(5) labeled cells, particularly on T(2)*WI (P<.05). Among pulse sequences, T(2)*WI demonstrated the highest DeltaSI (P<.05). CONCLUSION: The human umbilical cord blood MSCs can be labeled with Fe(2)O(3)-PLL without significant change in viability and apoptosis. The suspension of labeled MSCs can be imaged with standard 1.5-T MR equipment.     

Effect of vasodilator hydralazine on tumor microvascular random flow and blood volume as measured by intravoxel incoherent motion (IVIM) weighted MRI in conjunction with Gd-DTPA-Albumin enhanced MRI.

We studied the effect of hydralazine on tumor blood volume fraction and microvascular random flow velocity magnitude by IVIM weighted MRI in conjunction with dynamic Gd-DTPA-Albumin enhanced MRI. Blood volume fraction maps were obtained from the dynamic Gd-DTPA-Albumin enhanced MRI measurements. The average blood volume fraction of R3230 AC adenocarcinoma decreased from 0.125 +/- 0.022 (s.d.) ml/g to 0.105 +/- 0.018 (s.d.) ml/g (p < 0.001) after the administration of hydralazine at a dose of 5 mg/kg. The microvascular random flow velocity magnitude maps were obtained from the IVIM weighted MRI signals by utilizing the Gd-DTPA-Albumin measured blood volume fractions as an input in the compartmental modeling analysis of the IVIM weighted MRI signal. The random-directional microvascular flow induced MRI signal attenuation was separated from the molecular diffusion induced signal attenuation. Flow induced attenuation was more significant after the administration of hydralazine. The mean microvascular random flow velocity magnitude increased from 0.52 +/- 0.15 (s.d.) mm/sec to 0.73 +/- 0.23 (s.d.) mm/sec (p < 0.05) in the presence of the above blood volume fraction change.