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At least 325 records · Page 18Linked to original sources
PubMed2002

Primary acute myeloid leukaemia blasts resistant to cytokine-induced differentiation to dendritic-like leukaemia cells can be forced to differentiate by the addition of bryostatin-1.

Primary acute myeloid leukaemia (AML) blasts can be induced to differentiate into dendritic-like leukaemia cells (DLLC) by culture with certain cytokine combinations. DLLC offer potential for use as autologous vaccines based on their ability to present putative leukaemia-specific antigens to T cells. It has been reported, however, that in around 30-50% of AML cases the leukaemia cells are not capable of undergoing DLLC differentiation. The purpose of this study was to identify the features that represent successful DLLC differentiation and, for those cases shown to be resistant to cytokine-induced differentiation, to use differentiating agents in an attempt to overcome the differentiation block. Leukaemia cells derived from 42 patients with AML were cultured in vitro with cytokines GM-CSF, IL-4 and TNFalpha/CD40L. In 22 cases the leukaemic cells underwent DLLC differentiation based on characteristic morphological changes and expression of costimulatory and dendritic cell-associated molecules. Four cases were not evaluable because of poor viability over the culture period. The remaining 16 cases failed to show evidence of DLLC differentiation. Many of these differentiation resistant cases were associated with poor risk karyotypic features. Nine of the resistant cases were selected for further study. Differentiating agents trichostatin (TSA), azacytidine (AZA) and bryostatin (BRYO) were used in combination with cytokines for the first 96 h of the culture period. Bryostatin (BRYO) alone was shown to be capable of overcoming differentiation resistance and allowing DLLC differentiation to proceed.

Acute Disease↗
PubMed2004

Single-stranded DNA-binding protein facilitates gel-free analysis of polymerase chain reaction products in capillary electrophoresis.

It has been recently demonstrated that single-stranded DNA-binding protein (SSB) can facilitate quantitative analyses of DNA, RNA, and proteins in gel-free capillary electrophoresis (CE). Here, we report the application of SSB-mediated gel-free CE for analyses of polymerase chain reaction (PCR) products. The unique ability of SSB to bind ssDNA but not double-stranded DNA (dsDNA) allows efficient separation of three types of DNA molecules in the PCR reaction mixture: primers, products (amplified templates), and by-products, which originate from non-specific DNA hybridization. SSB-mediated gel-free CE analysis of PCR products combines simplicity, high sensitivity, and outstanding quantitative capabilities. The ability of the method to distinguish between products and by-products makes this method an indispensable tool in preparative PCR (e.g., in the development of nucleotide aptamers).

DNA Primers↗
PubMed1974

Chemically defined inducers of alkylsulphatases present in Pseudomonas C12B.

When Pseudomonas C12B is grown on nutrient broth to the stationary phase, cell extracts contain two secondary alkylsulphatases (S1 and S2) active towards potassium decan-5-yl sulphate but not towards potassium pentan-3-yl sulphate and one primary alkylsulphatase (P1) active towards sodium dodecan-1-yl sulphate (sodium dodecyl sulphate). When 10mm-sodium hexan-1-yl sulphate is included in the nutrient broth an additional primary alkylsulphatase (P2) is produced. The S1, S2, P1 and P2 enzymes are also present in extracts of cells grown on broth containing the commercial detergent Oronite, together with an additional secondary alkylsulphatase (S3) active towards pentan-3-yl sulphate as well as decan-5-yl sulphate. The P2 primary alkylsulphatase can be induced by a number of primary and secondary alkyl sulphate esters but the induction of the S3 enzyme appears to be a more specific and complex process. Studies on the ability of different fractions separated from Oronite to act as inducers suggest that the combination of a long-chain secondary alkyl sulphate(s) and a long-chain secondary alcohol(s) is responsible for the appearance of the S3 enzyme. Potassium hexadecan-2-yl sulphate or potassium tetradecan-2-yl sulphate, in combination with either hexadecan-2-ol or tetradecan-2-ol, can serve as inducers for the enzyme. Some characteristics of these specific inducer systems have been elucidated.

Cell Division↗
PubMed2001

CpG oligodeoxynucleotides with hepatitis B surface antigen (HBsAg) for vaccination in HBsAg-transgenic mice.

DNA motifs containing unmethylated CpG dinucleotides within the context of certain flanking sequences enhance both innate and antigen-specific immune responses, due in part to the enhanced production of Th1-type cytokines. Here we explored the ability of CpG-containing oligodeoxynucleotides combined with recombinant hepatitis B surface antigen (HBsAg) to induce Th1 responses in mice that are transgenic for this antigen and that represent a model for asymptomatic hepatitis B virus chronic carriers. This was compared to hepatitis B virus-specific DNA-mediated immunization, which we have previously shown to induce the clearance of the transgene expression product and the down-regulation of hepatitis B virus mRNA in this transgenic mouse lineage. In control nontransgenic C57BL/6 mice, three immunizations with HBsAg and CpG triggered the production of anti-HBs antibodies and of HBs-specific T cells that secrete gamma interferon but do not display any HBsAg-specific cytotoxic activity. In the HBsAg-transgenic mice, immunization with HBsAg and CpG oligodeoxynucleotides, but not with CpG alone, induced the clearance of HBsAg circulating in the sera, with a concomitant appearance of specific antibodies, and was able to regulate the hepatitis B virus mRNA constitutively expressed in the liver. Finally, adoptive transfer experiments with CD8(+) T cells primed in C57BL/6 mice with HBsAg and CpG oligodeoxynucleotide-based immunization show that these cells were able to partially control transgene expression in the liver and to clear the HBsAg from the sera of recipient transgenic mice without an antibody requirement. CpG oligodeoxynucleotides motifs combined with HBsAg could therefore represent a potential therapeutic approach with which to treat chronically infected patients.

Adjuvants, Immunologic↗
PubMed2002

Effect of combined electrostimulation and plyometric training on vertical jump height.

PURPOSE: This study investigated the influence of a 4-wk combined electromyostimulation (EMS) and plyometric training program on the vertical jump performance of 10 volleyball players. METHODS: Training sessions were carried out three times weekly. Each session consisted of three main parts: EMS of the knee extensor muscles (48 contractions), EMS of the plantar flexor muscles (30 contractions), and 50 plyometric jumps. Subjects were tested before (week 0), during (week 2), and after the training program (week 4), as well as once more after 2 wk of normal volleyball training (week 6). Different vertical jumps were carried out, as well as maximal voluntary contraction (MVC) of the knee extensor and plantar flexor muscles. RESULTS: At week 2, MVC significantly increased (+20% knee extensors, +13% plantar flexors) as compared to baseline ( < 0.05). After the 4-wk training program, different vertical jumps considered were also significantly higher compared to pretraining ( < 0.001), and relative gains were comprised between 8-10% (spike-counter movement jump) and 21% (squat jump). The significant increases in maximal strength and explosive strength produced by the present training program were subsequently maintained after an additional 2 wk of volleyball training. CONCLUSION: EMS combined with plyometric training has proven useful for the improvement of vertical jump ability in volleyball players. This combined training modality produced rapid increases (approximately 2 wk) of the knee extensors and plantar flexors maximal strength. These adaptations were then followed by an improvement in general and specific jumping ability, likely to affect performance on the court. In conclusion, when EMS resistance training is proposed for vertical jump development, specific work out (e.g., plyometric) must complement EMS sessions to obtain beneficial effects.

Adult↗
PubMed1989

Characterization and site-specific mutagenesis of the calcium-binding protein oncomodulin produced by recombinant bacteria.

A bacterial expression system for the parvalbumin-like calcium-binding protein oncomodulin has been constructed. This system can yield 50-fold more oncomodulin than the richest known mammalian source, the rat Morris hepatoma 5123. The bacterially produced protein folded correctly as monitored by UV, fluorescence, and 1H nuclear magnetic resonance spectroscopy and is immunologically identical to rat hepatoma oncomodulin. A calcium-specific conformational change is observed in the bacterial oncomodulin similar to that of the hepatoma protein. A modification of the putative calcium-specific CD loop by site-directed mutagenesis, which changed Asp-59 to Glu-59, eliminates calcium-specific effects. In contrast to the native molecule, the mutant Glu-59 now exhibits a magnesium-induced conformational change when monitored by UV difference or fluorescence excitation spectroscopy. The availability of large amounts of bacterially produced oncomodulin combined with the ability to modify at will the metal-binding ligands should now allow dissection of the unusual pairing in oncomodulin of one calcium-specific calmodulin-like site with one calcium/magnesium parvalbumin-like site.

Amino Acid Sequence↗
PubMed1992

New scoring system for prediction of ovarian malignancy based on transvaginal color Doppler sonography.

The goal of this prospective study was to develop a new scoring system using transvaginal color and pulsed Doppler characterization of ovarian lesions. Transvaginal color Doppler sonography was performed on 812 women, among whom 174 adnexal masses were found and analyzed. Ovarian lesions were assessed by means of morphological and color Doppler scoring systems. Tumors were characterized ultrasonically as benign or suspected of being malignant. Scoring system results were correlated with histopathological findings. Among our study group, 38 malignant and 136 benign ovarian tumors were found and verified. The color Doppler scoring system was very useful in distinguishing benign from malignant masses, with a sensitivity of 97.3% and a specificity of 100%, compared with the morphological scoring system's sensitivity of 92.1% and specificity of 94.8%. We also evaluated a combination of both scoring systems. We believe that new color Doppler scoring system maximizes the ability to discriminate between benign and malignant entities.

Adult↗
PubMed2005

Cell-based vaccines for renal cell carcinoma: genetically-engineered tumor cells and monocyte-derived dendritic cells.

Initial vaccine developments for renal cell carcinoma (RCC) have concentrated on cell-based approaches in which tumor cells themselves provide mixtures of unknown tumor-associated antigens as immunizing agents. Antigens derived from autologous tumors can direct responses to molecular composites characteristic of individual tumors, whereas antigens derived from allogeneic tumor cells must be commonly shared by RCC. Three types of cell-based vaccine for RCC have been investigated: isolated tumor cell suspensions, gene modified tumor cells and dendritic cells (DCs) expressing RCC-associated antigens. Approaches using genetic modification of autologous RCC have included ex vivo modification of tumor cells or modification of tumors in vivo. We have used gene-modification of allogeneic tumor cell lines to create generic RCC vaccines. More recently, emphasis has shifted to the use of DCs as cell-based vaccines for RCC. DCs have moved to a position of central interest because of their excellent stimulatory capacity, combined with their ability to process and present antigens to both naive CD4 and CD8 cells. The long impasse in identifying molecular targets for specific immunotherapy of RCC is now rapidly being overcome through the use of tools and information emerging from human genome research. Identification of candidate molecules expressed by RCC using cDNA arrays, combined with protein arrays and identification of peptides presented by MHC molecules, allow specific vaccines to be tailored to the antigenic profile of individual tumors, providing the basis for development of patient-specific vaccines.

Cancer Vaccines↗
PubMed1981

Uptake of radioiodine in follicles of dog C-cell complexes studied by autoradiograph and immunoperoxidase staining.

C-cell complexes are special cell groups consisting of a mass of C-cells associated with other epithelial elements and cysts. They are remnants of ultimobranchial bodies retaining fetal characteristics. In the C-cell complexes there are follicular cells in various stages of differentiation, i.e., the cell clusters not yet organized into follicles, primordial follicles with small lumens and comparatively enlarged follicles storing plentiful amounts of colloid. They have a morphology similar to follicular cells of fetal thyroid glands and react to antiserum to 19S thyroglobulin. In order to determine whether or not the follicles in these complexes have the ability to incorporate radioiodine, autoradiography after a single injection of 125I was combined with immunoperoxidase staining using specific anti-calcitonin, anti-C-thyroglobulin, and anti-19S thyroglobulin antisera. The 19S-positive cells not yet organized into follicles did not take up radioiodine. Primordial follicles showed a heavy accumulation of silver grains over their follicular lumens storing new 19S thyroglobulin as colloid. Comparatively enlarged follicles revealed a strong autoradiographic reaction and their labeling patterns were identical with those of typical thyroid follicles. These results confirm that the follicles in C-cell complexes, as well as thyroid follicles, can incorporate radioiodine and are related to thyroid hormone synthesis. That is, functional thyroid follicles can arise from the ultimobranchial bodies.

Animals↗
PubMed1989

Induction of antigen-specific immunity with monoclonal anti-idiotypic antibodies in vivo: differences in potency and comparison of immunochemical properties.

Anti-idiotypic (Id) antibodies provide a means other than antigen of clone-specific regulation of immune responses, and have been proposed as an alternative form of vaccine. However, the requirements for effective induction of immunity by anti-Id are not understood. Nine monoclonal anti-idiotope antibodies (anti-Id mAb) were derived in the Ia. 7 model system. While all nine anti-Id mAb induced comparable Ab3 responses in vivo as detected by ELISA, there were dramatic differences in the potency of the antigen-specific components of the responses induced by the nine anti-Id mAb. Anti-Id mAb that were indistinguishable in isotype, combining site relatedness, fine specificity on a panel of mAb, end point binding titers, competitive binding and ability to induce Ab3 differed dramatically in their ability to induce antigen-specific immunity in vivo, thus ruling out several models for explaining differences in induction.

Animals↗
PubMed1979

Procarbazine-induced specific-locus mutations in male mice.

Procarbazine is used in drug-combination treatment of Hodgkin's disease. The specific locus method was used to test and confirm the ability of procarbazine to induce gene mutations in pre- and post-meiotic germ cells of male mice. The lowest dose of procarbazine that significantly increased the mutation frequency in As spermatogonia over the control frequency was 400 mg/kg (P = 0.003). The corresponding dose for the post-spermatogonial germ-cell stages was 600 mg/kg (P = 0.009). The dose--response was linear for the point estimates of the mutation frequencies after treatment of As spermatogonia with 0, 200, 400 and 600 mg/kg. The point estimate of the mutation frequency at the 800 mg/kg level was one-third of that expected from a linear extrapolation. Variation in mutation rates among the 7 loci between the lowest (a locus) and the highest (p locus) was 12-fold. Only 24% of procarbazine-induced specific locus mutations in As spermatogonia were lethal in the homozygous condition. From the mutation spectra and the viability tests, it is concluded that procarbazine-induced mutations may be mainly due to base-pair changes. Procarbazine-induced specific-locus mutations fulfilled the criteria for the estimation of the doubling dose, the dose necessary to induce as many mutations as occur spontaneously. The doubling dose of procarbazine in As spermatogonia of mice was 114 mg/kg. The therapeutic dose for procarbazine is about 215 mg/kg. If man and mouse were equally sensitive, this dose would induce 1.9 times as many mutations as arise spontaneously. From the incidence of patients with Hodgkin's disease (1 : 42 000) the calculated population dose of procarbazine is 5.12 micrograms/kg. Assuming equal sensitivity between the sexes we can calculate, for an estimated number of 30 000 genes, the induction of about 22 mutations per million children due to procarbazine treatment. The same number of induced mutations can be calculated if the risk of patients is used for the estimation of the genetic hazard.

Animals↗
PubMed2004

Evidence of histamine receptors in fish brain using an in vivo [14C]2-deoxyglucose autoradiographic method and an in vitro receptor-binding autoradiographic method.

It was hypothesized that fish possess functioning H1 histamine receptors that have the ability to bind agonists and antagonists specific to the H1 histamine receptor subtype. For these experiments, a combination of a novel, in vivo 2-deoxyglucose method and a standard in vitro autoradiography procedure was utilized. A regional, statistically significant dose response in neurological functioning was observed when fish were exposed to histaminergic agents (i.e., H1 agonists and antagonists), which created the first neurological profile for the H1 histamine receptor in fish brain. The H1 histamine receptor was chosen as a characterization receptor in fish because histamine has been linked to a variety of neurological functions such as the control of arousal, attention, sensory processing, and cognition. Histamine also plays a role in pituitary hormone secretion, appetite control, and, potentially, regulation of vestigular reactivity. In addition, the fish brain is well characterized structurally, and the existence of an H3-like receptor has been documented recently in zebrafish. However, to date there is little detailed information about specific localization and functioning of the H1 histamine receptor in fish.

Animals↗
PubMed2004

Probabilistic cross-link analysis and experiment planning for high-throughput elucidation of protein structure.

Emerging high-throughput techniques for the characterization of protein and protein-complex structures yield noisy data with sparse information content, placing a significant burden on computation to properly interpret the experimental data. One such technique uses cross-linking (chemical or by cysteine oxidation) to confirm or select among proposed structural models (e.g., from fold recognition, ab initio prediction, or docking) by testing the consistency between cross-linking data and model geometry. This paper develops a probabilistic framework for analyzing the information content in cross-linking experiments, accounting for anticipated experimental error. This framework supports a mechanism for planning experiments to optimize the information gained. We evaluate potential experiment plans using explicit trade-offs among key properties of practical importance: discriminability, coverage, balance, ambiguity, and cost. We devise a greedy algorithm that considers those properties and, from a large number of combinatorial possibilities, rapidly selects sets of experiments expected to discriminate pairs of models efficiently. In an application to residue-specific chemical cross-linking, we demonstrate the ability of our approach to plan experiments effectively involving combinations of cross-linkers and introduced mutations. We also describe an experiment plan for the bacteriophage lambda Tfa chaperone protein in which we plan dicysteine mutants for discriminating threading models by disulfide formation. Preliminary results from a subset of the planned experiments are consistent and demonstrate the practicality of planning. Our methods provide the experimenter with a valuable tool (available from the authors) for understanding and optimizing cross-linking experiments.

Algorithms↗
PubMed2006

Three methionine residues located within the regulator of conductance for K+ (RCK) domains confer oxidative sensitivity to large-conductance Ca2+-activated K+ channels.

Methionine-directed oxidation of the human Slo1 potassium channel (hSlo1) shifts the half-activation voltage by -30 mV and markedly slows channel deactivation at low concentrations of intracellular Ca2+ ([Ca2+]i). We demonstrate here that the contemporaneous mutation of M536, M712 and M739 to leucine renders the channel functionally insensitive to methionine oxidation caused by the oxidant chloramine-T (Ch-T) without altering other functional characteristics. Coexpression with the auxiliary beta1 subunit fails to restore the full oxidative sensitivity to this triple mutant channel. The Ch-T effect is mediated specifically by M536, M712 and M739 because even small changes in this residue combination interfere with the ability to remove the oxidant sensitivity following mutation. Replacement of M712 or M739, but not M536, with the hydrophilic residue glutamate largely mimics oxidation of the channel and essentially removes the Ch-T sensitivity, suggesting that M712 and M739 may be part of a hydrophobic pocket disrupted by oxidation of non-polar methionine to the more hydrophilic methionine sulfoxide. The increase in wild-type hSlo1 open probability caused by methionine oxidation disappears at high [Ca2+]i and biophysical modelling of the Ch-T effect on steady-state activation implicates a decrease in the allosteric coupling between Ca2+ binding and the pore. The dramatic increase in open probability at low [Ca2+]i especially within the physiological voltage range suggests that oxidation of M536, M712 or M739 may enhance the Slo1 BK activity during conditions of oxidative stress, such as those associated with ischaemia-reperfusion and neurodegenerative disease, or in response to metabolic cues.

Cell Line↗
PubMed1995

SmaCS: smart classification system for the design, maintenance and use of complex terminologies. Application in pediatric cardiology.

The development of a generic Smart Classification System (SmaCS) for the design, construction, maintenance, and use of complex controlled terminologies is described. The ability of SmaCS to create and maintain terminologies that are combinations of structured terms and domain-specific knowledge is an important aspect of its design. SmaCS can therefore be used both for both controlled data-collection, using integrity rules included in the terminology, and intelligent data-retrieval. The design and implementation of SmaCS and application in the domain of pediatric cardiology are described.

Cardiology↗
PubMed2001

Augmenting transgene expression from carcinoembryonic antigen (CEA) promoter via a GAL4 gene regulatory system.

Though extensively studied, the use of tissue- or cell-type-specific promoters to target transgene expression is hampered by their weak activity. We hypothesized that this problem could be addressed by using a GAL4 gene regulatory system, wherein a weak, tissue-specific promoter would drive expression of the GAL4/VP16 fusion protein (GV16), which in turn would transactivate a minimal synthetic promoter, GAL4/TATA (GT), upstream of a transgene. To test this hypothesis, we constructed adenoviral vectors expressing a lacZ or GV16 gene driven by a carcinoembryonic antigen (CEA) promoter (Ad/CEA-LacZ or Ad/CEA-GV16) and evaluated levels of transgene expression they produced in cultured cells and in subcutaneous tumors after intratumoral administration. In CEA-positive cells, treatment with Ad/CEA-GV16 + Ad/GT-LacZ versus Ad/CEA-LacZ increased transgene expression 20- to 100-fold. In CEA-negative cells, treatment with Ad/CEA-GV16 + Ad/GT-LacZ increased transgene expression to a much lower degree (6- to 8-fold). In addition, analysis of Bax gene-mediated cell death revealed that this system can be used to avoid Bax's toxic effects on CEA-negative cells without compromising its ability to kill CEA-positive cells in vitro and in vivo. Thus, the combination of a tissue-specific promoter with the GAL4 gene regulatory system could be useful for targeting transgene expression.

Adenoviridae↗
PubMed1998

Detection of glaucoma with scanning laser polarimetry.

OBJECTIVE: To determine which retinal nerve fiber layer (RNFL) measures obtained with scanning laser polarimetry are most useful in detecting early to moderate glaucomatous visual field loss. SUBJECTS AND METHODS: One eye from 84 healthy individuals and 83 patients with early to moderate glaucomatous visual field loss (167 eyes) was assessed with a scanning laser polarimeter (Laser Diagnostic Technologies, San Diego, Calif). Three separate scans were obtained, and a baseline scan was created and used in the analyses. Integrated software (program GDx, version 1.0.02; Laser Diagnostic Technologies) was evaluated by assessing its sensitivity and specificity for detecting early and moderate glaucomatous visual field loss. Fisher linear discriminant functions also were developed in this population to assess sensitivity and specificity and were compared with the GDx analyses. RESULTS: There were statistically significant differences between the healthy and glaucomatous eyes for 14 of the 15 RNFL measures (P = .001). However, considerable overlap in measurements between groups was found. With the GDx number, the area under the receiver operator characteristic (ROC) curve was 0.78, and the sensitivity and specificity were 82% and 62%, respectively. Applying the best discriminant function using 3 variables (average thickness, ellipse modulation, and average ellipse thickness) to our study population resulted in an area under the ROC curve of 0.89 and a sensitivity and specificity of 74% and 92%, respectively. CONCLUSIONS: A combination of RNFL measures obtained using the scanning laser polarimeter improved the ability to differentiate between healthy eyes and eyes with early and moderate glaucomatous visual field loss. Analyses using GDx software did not differentiate between healthy and glaucomatous eyes as well as the discriminant analysis function did.

Aged↗
PubMed2002

Combined use of PRINS and FISH in the study of the dystrophin gene.

The efficacy of fluorescence in situ hybridization (FISH) may be limited in specific applications by low-resolution sensitivity. Primed in situ labeling (PRINS) is based on specific hybridization of an unlabeled oligonucleotide with a denatured template and synthesis of a single-strand DNA in situ. This method may represent a powerful alternative to FISH for gene mapping because of its ability to generate multiple independent signals within the same gene segment. We investigated the specificity of signals produced by a modified PRINS protocol combining a centromeric probe for the X-chromosome with specific primers for 3'- and 5'-terminal regions of the dystrophin gene. In approximately 70% of nuclei from male and female subjects, we detected one or two large signals (X-chromosome centromere) and two or four smaller signals (the two regions of the dystrophin gene). Specific hybridization of the oligonucleotides on Xp was demonstrated by localization of the smaller (dystrophin) and larger (X-centromere) signals on the same chromosome. Simultaneous hybridization with a centromeric probe and gene-specific oligonucleotides allowed localization of PRINS signals, and assessment of the specificity of the primers used for hybridization. This approach could facilitate identification of female carriers of small intragenic deletions in the dystrophin gene.

Base Sequence↗