Clone B1 Research Articles

Morphological, agronomical, physiological and molecular characterization of a high sugar mutant of sugarcane in comparison to mother variety.

Sugarcane is a significant crop plant with the capability of accumulating higher amount of sucrose. In the present study, a high sucrose content sugarcane mutant clone, GXB9, has been studied in comparison to the low sucrose mother clone B9 on morphological, agronomical and physiological level in order to scrutinize the variation because of mutation in GXB9 in field under normal environmental condition. The results showed that GXB9 has less germination, tillering rate, stalk height, leaf length, leaf width, leaf area, number of internodes, internode length and internode diameter than B9. Qualitative traits of leaf and stalk displayed significant variation between GXB9 and B9. Endogenous hormones quantity was also showed variation between the two clones. The relative SPAD reading and chlorophyll a, b concentrations also showed variation between GXB9 and B9. The photosynthetic parameter analysis indicated that the GXB9 has significantly higher net photosynthetic rate (Pn), stomatal conductance (gs) and transpiration rate (Tr) than B9. The qRT-PCR analysis of genes encoding enzymes like SPS, SuSy, CWIN, and CeS showed upregulation in GXB9 and downregulation in B9. However, these genes were significantly differentially expressed between the immature and maturing internodes of GXB9. The cane quality trait analysis showed that GXB9 had higher juice rate, juice gravity purity, brix, juice sucrose content and cane sucrose content than B9. The yield and component investigation results indicated that GXB9 had lower single stalk weight, however higher number of millable stalks per hectare than B9, and GXB9 had lower theoretical cane yield than B9. SSR marker analysis showed genetic variation between GXB9 and B9. This study has shown significant variation in the traits of GXB9 in comparison to B9 which advocates that GXB9 is a high sugar mutant clone of B9 and an elite source for future breeding.

Characterization of a new case of XMLV (Bxv1) contamination in the human cell line Hep2 (clone 2B)

The use of misidentified cell lines contaminated by other cell lines and/or microorganisms has generated much confusion in the scientific literature. Detailed characterization of such contaminations is therefore crucial to avoid misinterpretation and ensure robustness and reproducibility of research. Here we use DNA-seq data produced in our lab to first confirm that the Hep2 (clone 2B) cell line (Sigma-Aldrich catalog number: 85011412-1VL) is indistinguishable from the HeLa cell line by mapping integrations of the human papillomavirus 18 (HPV18) at their expected loci on chromosome 8. We then show that the cell line is also contaminated by a xenotropic murine leukemia virus (XMLV) that is nearly identical to the mouse Bxv1 provirus and we characterize one Bxv1 provirus, located in the second intron of the pseudouridylate synthase 1 (PUS1) gene. Using an RNA-seq dataset, we confirm the high expression of the E6 and E7 HPV18 oncogenes, show that the entire Bxv1 genome is moderately expressed, and retrieve a Bxv1 splicing event favouring expression of the env gene. Hep2 (clone 2B) is the fourth human cell line so far known to be contaminated by the Bxv1 XMLV. This contamination has to be taken into account when using the cell line in future experiments.

Characterization of γδ T-cells via flow cytometry.

Stervbo et al. report on the influences of ageing on a variety of peripheral innate and adaptive immune populations in two publications in the October issue of this journal (Stervbo et al. 2015a; Stervbo et al. 2015b). These articles surveyed peripheral blood mononuclear cell phenotypes from donors recruited in the cross-sectional Protective Immunity in Ageing (PRIMAGE) study. The focus of that study was the investigation of fundamental immunologic questions of the natural ageing process using seasonal influenza vaccination as a model (PRIMAGE 2015). Both papers also nicely combined phenotypic and functional assessment of age-related differences in major compartments of the cellular immune system in peripheral blood. Unlike many earlier studies, Stervbo et al. also focused on the underinvestigated γδ T cell compartment (Stervbo et al. 2015a). This is valuable because there are very few data on the impact of age on this conserved minor T cell subset. It is therefore especially important to be certain of the exactitude of the published results. However, we would like to sound a note of caution in this regard due to the use of the pan-γδ antibody clone B1, which may not be able to identify every γδ T cell when used in complex antibody panels applied in polychromatic flow cytometry. These issues were addressed by us in a carefully reviewed and validated antibody panel recently published in the journal Cytometry Part A’s “OMIP” series of standardized antibody panels (Wistuba-Hamprecht et al. 2014). A possible explanation could be steric hindrance effects because the epitopes recognized by anti-CD3 antibodies and the B1-clone might be in close proximity, which is not the case for the 11F2-clone that for example reliably detects the γδ T-cell compartment. In parallel to publication of these findings, we informed the manufacturers and one of the largest companies immediately revised their technical data sheet in accordance with these results (Becton Dickinson 2015). It might therefore be problematic to perform subset analysis on the basis of a B1-positive cell population. We felt that readers of Stervbo et al. 2015a, 2015b should be aware of these potential limitations.

Airborne particulate matter, hydroxyl radical production and cellular toxicity

Studies suggest that oxidative stress is a cause for the observed pathology in lung after exposures to air pollution. The aim of this study was to examine the effects of different airborne particulate matter (PM) on the survival of human bronchial epithelial cells immortalized with adenovirus-12 SV40 hybrid virus clone 2b (BEAS-2B cells, S6 subclone; BEAS). This was achieved by correlating cell survival directly with the flux of hydroxyl radicals (•OH) generated in a mixture of cells and particulates. A relatively new and highly sensitive fluorescence-based method was used to detect and quantify •OH. Two different toxicological methods were employed to test BEAS-2B cell survival in the presence of various types of airborne PM. Low •OH production was observed when this cell line was exposed to diesel particulate matter resulting in moderate toxicity. Urban dust (UD) which produced similar •OH flux in the presence of cells, resulted in protection from phosphate buffer saline toxicity, a required control. Amorphous silica which did not generate •OH radicals was the most toxic of all the particles tested. On the other hand, both cell death and hydroxyl radical formation were substantially enhanced when an external biological reductant, β-nicotinamide adenine dinucleotide phosphate, was added to a suspension of cells and UD. These results suggest that oxidants are crucial in generating high concentration of •OH and consequently cell death.

TCF7L2 splice variants have distinct effects on β-cell turnover and function

Type 2 diabetes manifests when the β-cell fails to secrete sufficient amounts of insulin to maintain normoglycemia and undergoes apoptosis. The disease progression results from an interplay of environmental factors and genetic predisposition. Polymorphisms in T-cell factor 7-like 2 (TCF7L2) strongly correlate with type 2 diabetes mellitus (T2DM). While TCF7L2 mRNA is upregulated in islets in diabetes, protein levels are downregulated. The loss of TCF7L2 induces impaired function and apoptosis. By analyzing human isolated islets, we provide three explanations for this opposite regulation and the mechanisms of TCF7L2 on β-cell function and survival. (i) We found TCF7L2 transcripts in the human β-cell, which had opposite effects on β-cell survival, function and Wnt signaling activation. While TCF7L2 clone B1, which lacks exons 13, 14, 15 and 16 induced β-cell apoptosis, impaired function and inhibited glucagon-like peptide 1 response and downstream targets of Wnt signaling, clones B3 and B7 which both contain exon 13, improved β-cell survival and function and activated Wnt signaling. (ii) TCF7L2 mRNA is extremely unstable and is rapidly degraded under pro-diabetic conditions and (iii) TCF7L2 depletion in islets induced activation of glycogen synthase kinase 3-β, but this was independent of endoplasmic reticulum stress. We demonstrated function-specific transcripts of TCF7L2, which possessed distinct physiological and pathophysiological effects on the β-cell. The presence of deleterious TCF7L2 splice variants may be a mechanism of β-cell failure in T2DM.

Frequency and Prognostic Impact of CD8 Expression In 5,523 Patients with Chronic Lymphocytic Leukemia (CLL) In Relation to Chromosomal Aberrations, IGHV Mutational Status and ZAP-70 Expression.

AbstractAbstract 4616Chronic lymphocytic leukemia (CLL) is an indolent lymphoma with largely heterogeneous clinical course. Identifying patients which benefit from early therapy is one of the most important issues in the management of CLL. Besides patient-specific characteristics and applicability of different treatment approaches the decision on therapy is based on conventional prognostic parameters as well as on chromosomal aberrations, IgVH mutational status and expression of ZAP-70, which all have been shown to be independently associated with outcome. The expression of CD8 has been rarely observed in CLL and its prognostic impact is unclear yet. In order to clarify the frequency of CD8 expression, its correlation to established prognostic parameters as well as its prognostic impact we analyzed a total of 5,523 patients with CLL by multiparameter flow cytometry including the expression of CD8 between August 2005 and August 2010. Fluorescence in situ hybridization (FISH) applying a standard set of probes for the detection of del(6q), del(11q22.3) (ATM), trisomy 12, del(13q14) (D13S25, D13S319), and del(17p13) (TP53) was performed in 3,407 patients. IGHV mutational status was determined and evaluable in 2,845 patients. Clinical follow-up data was available in 1,021 patients (median follow-up: 21.3 months). 61 patients (1.1%) showed an expression of CD8 (antibody clone B9.11, Immunotech, France) as compared to isotype used as negative control. CD8+ vs. CD8- cases did not differ in age (mean±SD: 70.3±9.7 vs. 68.3±10.4 years, n.s.) and the male/female ratio was 1.11 vs. 1.61 (n.s.). There were no significant differences in WBC count (mean±SD, 31.7±33.9 vs. 36.1±53.1 × 10e9/l) and hemoglobin level (Hb, mean±SD, 13.6±2.0 vs. 13.2±2.1 g/dl), while platelet counts were higher in CD8+ vs. CD8- cases (229±72 vs. 194±99 × 10e9/l, p=0.021). In CD8+ vs. CD8- cases chromosomal aberrations were detected by FISH analysis with the following frequencies: del(6q), 5.9% vs. 3.1%, n.s.; del(11q22.3), 17.1% vs. 10.7% (n.s.); trisomy 12, 20.0% vs. 14.5% (n.s.); del(13q14), 66.7% vs. 59.8% (n.s.); del(13q14) as sole chromosomal aberration, 41.2% vs. 45.5% (n.s.); del(17q13), 2.9% vs. 5.7% (n.s.). The IGHV status was mutated in 79.3% vs. 60.4% (p=0.054) in CD8+ vs. CD8- cases. Patients with CD8 expression had a significantly shorter time to therapy (TTT) as compared to CD8- patients (median TTT, 12.0 vs. 77.1 months, p=0.008). The following parameters showed a significant relation to a shorter TTT in univariate Cox analyses: CD8 positivity, p=0.011, relative risk (RR)=2.87; higher WBC count, p=0.008, RR=1.01 per 10 × 10e9 increase; del(11q22.3), p<0.001, RR=1.97; del(17p13), p=0.005, RR=1.72; higher % of CLL cells with ZAP-70 expression (antibody clone SBZAP, Immunotech, France), p=0.011, RR=1.04 per 10% increase. Parameters significantly related to a longer TTT in univariate Cox analyses were: higher Hb level, p<0.001, RR=0.86 per 1 g/dl; higher platelet count, p=0.021, RR=0.98 per 10 × 10e9 increase; and del(13q14) as sole chromosomal aberration, p<0.001, RR=0.58. Multivariate analysis identified two parameters to be independently related to a shorter TTT: higher ZAP-70 expression (p=0.002) and CD8 positivity (p=0.036), while a higher Hb level (p<0.001) and del(13q14) as sole chromosomal aberration (p=0.011) were identified to be independently related to a longer TTT. This data supports the further evaluation of the prognostic impact of CD8 expression in CLL in order to define its role in identifying the most appropriate timepoint for therapy and the most appropriate treatment modality for patients with CLL. Disclosures:Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

ESCs Require PRC2 to Direct the Successful Reprogramming of Differentiated Cells toward Pluripotency

Embryonic stem cells (ESCs) are pluripotent, self-renewing, and have the ability to reprogram differentiated cell types to pluripotency upon cellular fusion. Polycomb-group (PcG) proteins are important for restraining the inappropriate expression of lineage-specifying factors in ESCs. To investigate whether PcG proteins are required for establishing, rather than maintaining, the pluripotent state, we compared the ability of wild-type, PRC1-, and PRC2-depleted ESCs to reprogram human lymphocytes. We show that ESCs lacking either PRC1 or PRC2 are unable to successfully reprogram B cells toward pluripotency. This defect is a direct consequence of the lack of PcG activity because it could be efficiently rescued by reconstituting PRC2 activity in PRC2-deficient ESCs. Surprisingly, the failure of PRC2-deficient ESCs to reprogram somatic cells is functionally dominant, demonstrating a critical requirement for PcG proteins in the chromatin-remodeling events required for the direct conversion of differentiated cells toward pluripotency.

Molecular characteristics and functional analysis of full-length hepatitis B virus quasispecies from a patient with chronic hepatitis B virus infection

It has been hypothesized that naturally occurring mutations in HBV may play a role in the pathogenesis of HBV-related disease. We determined the molecular characteristics of naturally occurring HBV isolates and performed a functional analysis of full-length hepatitis B virus quasispecies from a patient with chronic hepatitis B. The 10 HBV clones isolated were identified as HBV genotype B4 and subtype adw. In most clones, amino acid substitutions and nucleotide changes occurred in a specific region of the core protein, X protein and in the core promoter. In the core protein, cI3L, cL60M, and cI97L were detected in 8 of 10 clones and cP130T was detected in all 10 clones. In the X protein, xI127M was detected in 5 clones. In the basal core promoter, the A1762T/G1764A mutation was found only in 1 clone. An 11-bp nucleotide insertion between nucleotides 1772 and 1773, was found in 2 clones. Six clones that were replication-competent exhibited variation in the level of replicating capacity in vitro even though the average genetic distance between the HBV clones was only 0.5% (range: 0.3–0.7%). Among the replication-competent 6 clones, 5 clones showed higher replication competency compared with clone B9 (reference clone used in this study) in Huh7 cells. However, 4 clones showed lower replication competency compared with clone B9 in HepG2 cells. In conclusion, the HBV virus exhibits genetic variation in the form of quasispecies with different mutation patterns, and these quasispecies may be recognized by distinct viral replication patterns even in patients with subtle genetic mutation.

Quantification of Circulating Regulatory T Cells by Flow Cytometry in Kidney Transplant Patients After Basiliximab Induction Therapy

Basiliximab is specific for CD25 and, therefore, can potentially affect the size and function of the CD25+ regulatory T cells (Tregs) pool in transplant recipients. Several recent reports have indeed suggested that this monoclonal antibody (mAb) decreased the number of circulating Tregs (1,2) but did not impair their suppressive function (1–3). Irrespective of its biologic effects, basiliximab can also hinder the detection of CD25 by certain flurochrome-conjugated antibodies, which bind a common CD25 epitope (4). Tregs depletion can, thus, artifactually be accentuated if a competing anti-CD25 clone is selected for flow cytometry detection. We have examined the most commonly available anti-CD25 clones regarding a possible epitopic competition with basiliximab and determined which of them can be used to rigorously monitor Tregs in basiliximab-treated transplant patients. An in vitro competition assay between basiliximab and eight different fluorochrome-conjugated anti-CD25 clones was carried out under the following conditions: immunomagnetically selected CD4+ cells (Miltenyi Biotec, Bergisch Gladbach, Germany) obtained from five healthy volunteers (after informed consent and with the approval of the local ethic committee) were activated for 2 days with 5 μg/mL of plate-coated anti-CD3 mAb and 2 μg/mL of soluble anti-CD28 mAb, then colabeled with fluorochrome conjugated anti-CD4 and anti-CD25 mAbs, alone or in the presence of increasing doses of basiliximab (basiliximab final concentrations ranging from 8×10−4 to 4×10−2 μg/mL). Proportion of CD25+ cells detected by flow cytometry (FACS Canto II, BD Biosciences, san Jose, CA) was analyzed for each anti-CD25 clones using FACS DIVA Software Version 2.0 (BD Biosciences). The eight anti-CD25 clones we tested were: B1 49.9 (Beckman Coulter, Fullerton, CA), ACT1 (Dako, Glostrup, Denmark), Bb10 and BF2 (Diaclone, Besançon, France), 2A3 (BD Biosciences), BC96 (eBioscience, San Diego, CA), MA251 (BD Biosciences), and 4E3 (Miltenyi Biotec). Two of these eight clones did not interfere with basiliximab and were able to correctly stain the CD25+ population regardless the concentration of basiliximab in which the cells have been preincubated (Fig. 1a). Accordingly, only these two clones, MA251 and 4E3, could detect the CD4+CD25+CD127− Tregs population in five basiliximab-treated transplant patients 1 month posttransplant (Fig. 1b).FIGURE 1.: Anti-CD25 clones MA251 and 4E3 can detect CD25+ cells in the presence of basiliximab. (a) CD25 expression on activated CD4+ T cells from a healthy volunteer was evaluated with eight different anti-CD25 fluorescent monoclonal antibodies (mAbs) (clones B1 49.9, ACT1, Bb10, BF2, 2A3, BC96, MA251, and 4E3) in the absence or the presence of basiliximab used at different concentrations. Percentages are the proportion of CD25+ cells among the CD4+ population. Similar results were observed with five different healthy volunteer. (b) Peripheral blood mononuclear cells from a transplant patient having received an induction therapy with basiliximab (20 mg intravenously at days 0 and 4) were obtained 1 month posttransplant and colabeled with the three CD4/CD25/CD127 fluorescent antibodies. The ability of eight different anti-CD25 fluorescent mAbs (clones B1 49.9, ACT1, Bb10, BF2, 2A3, BC96, MA251, and 4E3) to detect the CD4+CD25+CD127− regulatory T-cell subset (6,7) was compared. The direct comparison between the competing B1 49.9 clone with the noncompeting MA251 clone is shown. (Similar data to those obtained for clone B1 49.9 were observed for clones ACT1, Bb10, BF2, 2A3, and BC96; similar data to those obtained for clone MA251 were observed for clone 4E3; data shown are representative of data obtained from five different transplant patients.)At present, monitoring the Treg population is rather considered as a promising, albeit not yet validated, tool to guide immunosuppressive minimizing or weaning protocols after the initial period of transplantation, a period of time during which basiliximab is no longer detectable in serum. However, it is probably also informative to numerate Tregs during the earliest phase of transplantation when the relative expansion between the pools of regulatory and effector T cells might predict the outcome of the graft (5). Basiliximab interferes with the vast majority of commercially available anti-CD25 clones. We observed this interference within the 3 months after basiliximab treatment (data not shown). In the context of basiliximab induction therapy, intracellular forkhead box P3 staining should be privileged to properly detect the Tregs subset. When not available, a careful selection of the anti-CD25 clone used for flow cytometry is critical. Clones MA251 and 4E3 do not compete for a common epitope with basiliximab and can, thus, safely be used in this specific situation. ACKNOWLEDGMENTS The authors thank Solène MICHEL for her excellent technical assistance and Clémence MARIAT for her help in editing the manuscript. Farida Abadja Laboratoire de Néphrologie Université Jean Monnet France Eric Alamartine François Berthoux Christophe Mariat Laboratoire de Néphrologie Université Jean Monnet France Service de Néphrologie Dialyse et Transplantation rénale CHU de Saint-Etienne France Christian Genin Claude Lambert Laboratoire d'Immunologie Université Jean Monnet France

Inhibition of Cryptosporidium parvum infection of a mammalian cell culture by recombinant scFv antibodies

Two phage display antibody libraries (Tomlinson I and J) were screened against the whole oocysts of Cryptosporidium parvum to select for scFv (single chain variable fragment) antibodies. Three scFv antibodies were selected that bound to C. parvum oocysts as determined by monoclonal phage ELISA. DNA sequencing revealed that clone A11 lacked the majority of its V (H) chain. Clone B10 had a stop codon in the first framework region of the V (H) chain. We changed this stop codon to Gly by site-directed mutagenesis, and designated the variant mutB10. Clone B9 had a complete scFv gene with no internal stop codons. These antibody genes were individually subcloned into the pET-20b expression vector for soluble scFv antibody production. C. parvum infectivity was determined by infection of HCT-8 tissue culture monolayers and quantified by the foci detection method. By incubating C. parvum oocysts with individual scFv antibodies for 1 h at 37 degrees C prior to infecting the HCT-8 cells with the oocyst-scFv mixture, the infectivity of C. parvum was reduced in a dose-dependent manner. At the highest soluble scFv concentration tested (4 nmol), the mean number of infectious foci was reduced by 82%, 73% and 94% for the A11, B9 and mutB10 scFv, respectively. This inhibition of oocyst infectivity was abolished when the scFvs were exposed to boiling water. The results showed that the 3 selected scFvs bound to C. parvum oocysts, and their ability to neutralize infectivity may have potential therapeutic potential against cryptosporidiosis.

Characterization of effector functions of human peptide-specific CD4+ T-cell clones for an intracellular pathogen

CD4+ T cells are believed to play a dominant role in human defenses against Mycobacterium tuberculosis through production of interferon (IFN)-gamma, cytolytic T-cell (CTL) activity, and inhibition of intracellular mycobacterial growth. Most functional studies of CD4+ cells have used bulk T-cells that recognize crude mycobacterial antigens, and the functional capacity of individual human T cells is not well defined. We studied the functional capacity of human CD4+ T-cell clones that recognize a specific mycobacterial peptide. Clone B9 produced high concentrations of IFN-gamma and exhibited potent CTL activity, whereas clone D3 produced IFN-gamma but showed poor CTL activity. The CTL activity of clone B9 was inhibited by SrCl(2) and concanamycin A but not by anti-Fas antibodies. Clone B9 also reduced the mycobacterial burden in dendritic cells by more than 90%, and this antimycobacterial activity was inhibited by SrCl(2) and concanamycin A. We conclude that: (1) individual human peptide-specific CD4+ T-cell clones have differential capacity to produce Th1 cytokines and to lyse M tuberculosis-infected target cells; and (2) both granulysin and perforin contribute to the capacity of human CD4+ T-cells to lyse infected targets and to inhibit intracellular mycobacterial growth.

A bioluminescence assay for thyrotropin receptor antibodies predicts serum thyroid hormone levels in patients with de novo Graves’ disease

TSH receptor antibodies (TBII) in Graves' disease (GD) may be TSH receptor stimulating (TSAb) and blocking (TBAb) antibodies. In commercially available assays however, only total TBII titres can be measured, without discriminating between TSAb and TBAb. To design a TBII bioassay to detect of TSAb and to correlate TSAb activity with severity of hyperthyroidism in de novo GD patients. Thirty-five patients with de novo GD and 27 controls. The JP-26-26 cell line, which constitutively expresses the TSH receptor (TSHR), was stably transfected with a cyclic adenosine monophosphate responsive element--luciferase construct. The clone B1 exhibited a near linear increase in luminescence from 0.2 mU/l to 50 mU/l bovine TSH and was used as a TBII bioassay. TBII, free T4 and TSH were measured in the sera of all patients and controls. In the sera of 35 GD patients, TBII titres did not correlate with serum free T4 concentrations. In contrast, a strong and highly significant correlation was found between TSHR stimulating activity (luminescence) as measured with the TBII bioassay and serum free T4 levels (R = 0.80, P < 0.001). Interestingly, the luminescence/TBII ratio had a wide range at low TBII titres, whereas high TBII titres were associated with a low degree of TSHR activation. The TBII bioassay also detected TBAb in GD patients who spontaneously developed hypothyroidism. The B1-TBII-bioassay as developed in our laboratory has a high sensitivity for the detection of TSAb in GD and predicts the severity of hyperthyroidism in untreated GD patients. In addition, we found that high TBII titres are associated with weak TSHR activation.

68 REPRODUCTIVE PERFORMANCE OF CLONED BULLS

The generation of cloned animals by somatic cell nuclear transfer has been reported in a number of countries worldwide. However, progress has been impeded by the extremely low efficiency of cloning and health of some of the cloned animals. Surprisingly, little is known of the reproductive performance of viable clones when compared to the original cell donor, given that a major motivation of cloning is dissemination of superior genotypes. The aim of this study was to compare semen collected from three cloned bulls (Clones A1, B1, and B2) to that of the original donor bulls (Donors A and B). Parameters examined included ejaculate volume, sperm concentration and the motility characteristics of frozen/thawed semen using computed-assisted semen analysis (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA). The fertilization ability of each semen sample was examined using in vitro matured oocytes derived from abattoir-source ovaries. Frozen/thawed semen samples from donor and cloned bulls were prepared on a Percoll gradient and diluted with fertilization medium to a concentration of 1 million sperm/mL prior to fertilization (IVF). The number of blastocysts and total cell counts were analyzed on Day 7 of culture. Finally, in vitro-fertilized blastocysts (Day 7) were transfer to synchronized recipients (n = 49) to examine in vivo development. Proportional data for the in vitro development of embryos and subsequent pregnancy rates were analyzed by chi-square test, and embryo cell numbers were analyzed using Student's t-test. Progressive motility percentage between donor and cloned bull did not differ: Donor A (62.25 ± 3.89, n = 12); Donor B (66.69 ± 4.47, n = 13); Clone A1 (71.37 ± 8.57, n = 8); Clone B1 (73.75 ± 2.42, n = 12); Clone B2 (72.41 ± 3.26, n = 12). No obvious differences in kinetic motility parameters were evident between cloned and non-cloned donor animals. However, blastocyst rates were significantly higher in cloned bulls (Clone A1: 30.9%, 81/262; Clone B1: 34.4%, 98/285; and Clone B2: 42.9%, 120/280) compared to donor bulls (Donor A: 20.7%, 54/261; Donor B: 20.9%, 76/364). Total embryo cell numbers did not differ significantly between donor bulls (Donor A: 138.3 ± 5.3, n = 39; Donor B: 133.2 ± 5.2, n = 47) and cloned bulls (Clone A1; 126.3 ± 4.4, n = 45; Clone B1: 134.4 ± 7.1, n = 26; and Cloned B2: 140.1 ± 3.9, n = 46). Initial pregnancy rates on Day 30 were also not different between Donor A (42.3%, 11/26) and Clone A1 (47.8%, 11/23). Preliminary observations from the small data set on postpubertal cloned bulls indicate that semen production, semen morphology, and reproductive performance (in vitro and in vivo) were similar in terms of semen characteristics and reproductive performances when compared to their original donor bulls.

Melanoma heterogeneity: differential, invasive, metastatic properties and profiles of cathepsin B, D and L activities in subclones of the B16F10-NEX2 cell line

Tumour cell lines and in vivo growing tumours are heterogeneous, comprising different cell clones. To understand why some cells primarily invade a tissue, while others are more apt to metastasize, several clones from the established B16F10-Nex2 cell line were isolated and 10 viable cells of each clone were injected intravenously into C57Bl/6 and Balb/c mice. Two cell clones (Nex2B and Nex2D) showed contrasting metastatic abilities. Clone 2D rather than clone 2B colonized the lungs of both mice after intravenous injection. Surprisingly, clone 2B grew more rapidly than 2D after subcutaneous implantation, significantly reducing the survival of injected mice. Clearly, dissociation between subcutaneous growth and metastatic ability was observed in clones from the same tumour cell lineage. Clone Nex2B continuously released proteolytic activity, including cathepsin B, and showed a greater capacity to invade Matrigel than clone Nex2D. Clone Nex2D accumulated cathepsins B, D and L intracellularly and released a moderate proteolytic activity in vitro that was inhibited with the time of incubation. E-64-treated Nex2B cells injected subcutaneously showed a significant delay in tumour development and increased survival of challenged animals. A similar result was obtained on treatment of clone 2B with chagasin, a cysteine proteinase inhibitor from Trypanosoma cruzi, even at 2 microM. Clone Nex2D was less sensitive to pretreatment with inhibitors of cysteine proteases for tumour development in vivo. Our results suggest that, in a tumour cell population, cells dissociate into metastatic and non-metastatic subtypes, and that release or accumulation of cathepsins can be a differential trait of these cells.

439. Neuronal Membrane Specific Binding Peptides Isolated Via Phage Display Biopanning Against Trisialogangliosides

Background: The tropism of adeno-associated viral vectors can be altered by inserting peptides into the vector's coat. However, large peptides may alter the capacity of VP proteins to package DNA. In order to enhance the neural affinity and specificity of AAV binding, we have attempted to isolate short peptides which when inserted into the cap gene of the AAV genome, would retarget the vector toward neuronal membranes without hindering its transduction efficiency. We have previously reported a phage display biopanning strategy for the isolation of such peptides (Liu et al Mol Ther: 7(5) S351, 2003). We used trisialogangliosides (GT1b), a known receptor of Clostridial tetanus toxin present on neuronal membranes, as our target. Tetanus toxin owes its high neuronal affinity to specific GT1b binding providing an ideal interaction to replicate in AAV. Using a random 12 amino acid peptide phage library, we conducted a novel four round biopanning process against immobilized GT1b. In the final three rounds of biopanning, we eluted with recombinant Tetanus Toxin C Fragment (rTTC) to recover phage that specifically mimic its binding to GT1b. Four candidate peptides were enriched following the biopanning process. We selected the most common peptide (Clone 2A) which was present in 70% of the sequenced phage clones along with a second peptide (Clone 2B) that shared similar homology with Clone 2A (Liu et al Mol Ther: 7(5) S351, 2003).

Interaction of Trypanosoma rangeli Tejera, 1920 with different cell lines in vitro.

Intracellular multiplication of Trypanosoma rangeli was evaluated in vitro using experimental infection of Vero cells, murine macrophages, and promonocytes with T. rangeli Choachi, Macias, and SC-58 clone B1 strains. Our results revealed a low infectivity of all T. rangeli strains to these cell lines. Macrophages showed the highest infection rate; however, intracellular forms were no longer observed 48 h post infection Despite the observation of intracellular parasites up to 144 h post infection, the infection rates of Vero cells and J774G.8 promonocytes with these parasite strains were always below 5%. Pre-incubation of parasites with normal mouse serum increased the initial infectivity but not the time course of the infection. Under our experimental conditions, we did not observe any evidence of intracellular multiplication of T. rangeli within these cell lines.

Characterization of murine Th1 clones specific to egg antigen of Schistosoma japonicum and their interaction with cytokines.

T cell clones (B1, B21, B7, A25) specific to the soluble egg antigen (SEA) of Schistosoma japonicum were established from C3H/He mice immunized with SEA. These clones belonged to CD3+, CD4+ and CD8-Th1 cells, showing TCR-gamma delta-, TCR-alpha beta+ and Vbeta10b+. The molecular weights of target antigens recognized by the clones ranged from 51 to 80 kDa. Interleukin-2 (IL-2) and IL-12 could vigorously increase the proliferation response of the T clones to SEA; while IL-10 and transforming growth factor-beta1 (TGF-beta1) strongly inhibited the response. IL-12 activity was detected in the culture supernatant of T clones stimulated with SEA in the presence of APC (antigen presenting cells). This stimulation also upregulated the expression of the IL-12 receptor on the T clones. IL-12 from APC served as a costimulatory factor for the SEA induced proliferation of the T clone cells. Clone B1 was able to induce granuloma formation both in vivo and in vitro. These data provide further insight into the complicated interaction among SEA, T cell and cytokine at a clonal level in S. japonicum infection.

A link between cell cycle and cell death: Bax and Bcl-2 modulate Cdk2 activation during thymocyte apoptosis.

Resting thymocytes undergoing apoptosis in response to specific stimuli degrade the cdk inhibitor p27(Kip1) and upregulate Cdk2 kinase activity. Inhibition of Cdk2 kinase activity efficiently blocks cell death via certain apoptosis pathways whereas overexpression of Cdk2 accelerates such cell death, suggesting its involvement in the signal transduction pathways activated by certain apoptotic stimuli. We found that Cdk2 activation during thymocyte apoptosis can be regulated by p53, Bax and Bcl-2. The highly elevated Cdk2 kinase activity in the apoptosing thymocytes is not associated with its canonical cyclins, cyclin E and cyclin A, and requires de novo synthesis of proteins for activation to take place. We therefore propose Cdk2 activation to be a crucial event in distinct pathways of apoptosis and the point at which the cell cycle and cell death pathways interact.

Protein Kinase C-α Modulates Lipopolysaccharide-induced Functions in a Murine Macrophage Cell Line

Lipopolysaccharide (LPS), a potent modulator of macrophage functional activity, binds to CD14 and triggers the activation of several protein kinases, leading to the secretion of variety of immunomodulatory molecules such as nitric oxide and proinflammatory cytokines. In this study, we have examined the role of the alpha isoenzyme of protein kinase C (PKC) in the regulation of LPS-initiated signal transduction in macrophages. To this end, we have stably overexpressed a dominant-negative (DN) version of PKC-alpha (DN PKC-alpha) in the murine macrophage cell line RAW 264. 7. Clones overexpressing DN PKC-alpha were indistinguishable from the parental line with respect to morphology and growth characteristics. At the functional level, DN PKC-alpha overexpression strongly inhibited LPS-induced interleukin-1alpha mRNA accumulation, and to a lesser extent inducible nitric oxide synthase and tumor necrosis factor-alpha expression. DN-PKC-alpha overexpression did not cause a general unresponsiveness to LPS, as secretion of the matrix metalloproteinase-9 was up-regulated in our DN PKC-alpha-overexpressing clones. Moreover, LPS-induced phosphorylation and degradation of IkappaBalpha, NF-kappaB activation, as well as p38 mitogen-activated protein kinase and Jun N-terminal kinase phosphorylation, were not affected by DN PKC-alpha overexpression. Collectively, these data provide evidence that PKC-alpha regulates selective LPS-induced macrophage functions involved in host defense and inflammation.

High forskolin production in hairy roots of Coleus forskohlii.

Hairy roots of Coleus forskohlii were induced by infection with the Agrobacterium rhizogenes MAFF 03-01724 strain. Growth and forskolin production of two hairy root clones cultured in various liquid media were examined. Hairy root clone B9 grew well in woody plant liquid medium and showed a high forskolin yield (ca. 1.3 mg/ 100 ml flask) after 5 weeks of culture. The time course of growth and forskolin production of the clone B9 cultured in woody plant liquid medium was also examined. Rapid growth started at week 2 and continued until week 5. The highest forskolin yield (ca. 1.6 mg/100 ml flask) was obtained at week 5. Productivity was much higher than that previously reported.