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PubMed2006

Site discrimination in mixed-alkali glasses studied by cross-polarization NMR.

Cation-cation interactions are thought to play a significant role in shaping the nonlinear compositional dependence of ionic conductivity, known as the mixed-alkali effect (MAE) in glassy solid electrolytes. For providing a structural rationale of this effect, the discrimination of various cation sites in mixed-alkali glasses is of interest. In the present study, cross-polarization (CP) experiments have been applied to glasses in the system [(Li(2)O)x(Na(2)O)(1-x)](0.3)[B(2)O(3)]0.7 to discriminate between alkali ions by virtue of different heteronuclear (7)Li-(23)Na dipole-dipole coupling strengths. Cross-polarization studies involving two types of quadrupolar nuclei (both (7)Li and (23)Na have a spin-quantum number I = 3/2) are complicated by spin state mixing under radio frequency irradiation and magic-angle spinning (MAS). Therefore careful validation and optimization protocols are reported for the model compound LiNaSO(4) prior to conducting the measurements on the glassy samples. (23)Na -->( 7)Li CP/MAS NMR spectra have been obtained on glasses containing the Na(+) ions as the dilute species. They reveal that those lithium species interacting particularly strongly with sodium ions have the same average (7)Li chemical shift as the entire lithium population; the symmetrical situation applies to the (23)Na nuclei at the sodium rich end of the composition range. On the other hand, a clear site discrimination is afforded by temperature-dependent static (23)Na -->( 7)Li CP experiments, indicating that the Li ions that are most strongly interacting with sodium ions are strongly immobilized. This finding provides the first direct experimental evidence for the proposed secondary mismatch concept invoked for explaining the strong MAE in the dilute foreign ion limit.

Journal Article↗

PubMed2002

Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.).

Procedures for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) have been developed for chickpea (Cicer arietinum). Suspensions of intact chromosomes were prepared from root tips treated to achieve a high degree of metaphase synchrony. The optimal protocol consisted of a treatment of roots with 2 mmol/L hydroxyurea for 18 h, a 4.5-h recovery in hydroxyurea-free medium, 2 h incubation with 10 micromol/L oryzalin, and ice-water treatment overnight. This procedure resulted in an average metaphase index of 47%. Synchronized root tips were fixed in 2% formaldehyde for 20 min, and chromosome suspensions prepared by mechanical homogenization of fixed root tips. More than 4 x 10(5) morphologically intact chromosomes could be isolated from 15 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing eight peaks, representing individual chromosomes and/or groups of chromosomes with a similar relative DNA content. Five peaks could be assigned to individual chromosomes (A, B, C, G, H). The parity of sorted chromosome fractions was high, and chromosomes B and H could be sorted with 100% purity. PCR on flow-sorted chromosome fractions with primers for sequence-tagged microsatellite site (STMS) markers permitted assignment of the genetic linkage group LG8 to the smallest chickpea chromosome H. This study extends the number of legume species for which flow cytogenetics is available, and demonstrates the potential of flow cytogenetics for genome mapping in chickpea.

Cell Cycle↗

PubMed2004

Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics.

We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative. We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures. Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis. We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S. cerevisiae and P. pastoris on a scale of 5 to 10 mg. Both batch and fed batch fermentation methods were applied to S. cerevisiae. The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins. We carried out only fed batch fermentations on P. pastoris strains. Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression. The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag. They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography. This was followed by gel filtration for further purification and for buffer exchange. This three-step purification procedure is necessary to obtain highly purified proteins from yeast. The purified proteins have successfully been subjected to crystallization and biophysical analysis.

Chromatography, Affinity↗

PubMed2005

Genome sequencing in microfabricated high-density picolitre reactors.

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Electrophoresis, Capillary↗

PubMed1996

A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells.

An optimized protocol has been developed for the efficient and rapid genetic modification of sugar beet (Beta vulgaris L.). A polyethylene glycol-mediated DNA transformation technique could be applied to protoplast populations enriched specifically for a single totipotent cell type derived from stomatal guard cells, to achieve high transformation frequencies. Bialaphos resistance, conferred by the pat gene, produced a highly efficient selection system. The majority of plants were obtained within 8 to 9 weeks and were appropriate for plant breeding purposes. All were resistant to glufosinate-ammonium-based herbicides. Detailed genomic characterization has verified transgene integration, and progeny analysis showed Mendelian inheritance.

Biotechnology↗

PubMed2026

Cryopreservable dopaminergic progenitors derived from human iPSCs with accelerated loss of pluripotency and early functional restoration in Parkinsonian rats.

Midbrain dopaminergic progenitors (mDAp) derived from human pluripotent stem cells have demonstrated promising safety and efficacy in Phase I clinical transplantation trials for Parkinson's disease (PD). To further improve the translational potential of this approach, strategies that accelerate the loss of pluripotency, increase the yield of in vitro mDA progenitors, and promote post-transplantation neurite outgrowth may be beneficial. Here, we developed an optimized protocol building upon our previously established neural induction method, with further refinements, to efficiently convert induced pluripotent stem cells (iPSCs) into mDA neurons. Additionally, we applied n-butylidenephthalide, which selectively reduced pluripotency-associated gene expression and enhanced neurite outgrowth during differentiation. With these improved techniques, 71% of iPSCs differentiated into mDA neurons, showing burst dopamine secretion and phasic electrophysiological activities with external stimuli. To evaluate the safety, cryopreserved mDAp were transplanted into immunodeficient mice. No teratoma or neural tumor was observed within 24 weeks post-implantation. In 6-OHDA PD rats, mDAp survived and differentiated into mDA neurons in the host striatum within eight weeks post-transplantation, leading to significant functional recovery. The current differentiation process, therefore, enables the generation of cryopreservable, off-the-shelf mDAp, with accelerated loss of the pluripotency marker OCT4 in vitro and early functional recovery following transplantation.

Journal Article↗

PubMed2005

The value of immunohistochemistry in sentinel lymph node histopathology in breast cancer.

The optimal protocol for the histopathological examination of sentinel lymph nodes (SLNs) in breast cancer has not been determined. The value of more detailed examination using immunohistochemistry (IHC) is controversial. A total of 476 SLNs from 216 patients were reviewed. Sentinel lymph nodes were sectioned at three levels at 100 mum intervals and stained with haematoxylin and eosin (H&E). If the H&E sections showed no evidence of metastasis, then the three serial sections were stained with a murine monoclonal anti-cytokeratin antibody (CAM 5.2). Metastatic deposits were classified as macrometastasis (> 2.0 mm), micrometastasis (0.2-2.0 mm) or isolated tumour cells (ITC, < 0.2 mm). Of the 216 patients, 56 (26%) had metastasis as identified by H&E. Immunohistochemistry detected metastatic deposits in a further nine patients (4%), of whom four (2%) had micrometastasis and five (2%) had ITC only. Those cases with micrometastases were all, on review, visible on the H&E sections. Immunohistochemistry detects only a small proportion of metastasis in SLNs. All metastatic deposits identified by IHC were either micrometastasis or ITC. Until the prognostic significance of these deposits has been determined, IHC may be of limited value in the histopathological examination of SLNs.

Axilla↗

PubMed2002

The impact of antithymocyte globulin on short-term toxicity after allogeneic stem cell transplantation.

Antithymocyte globulin (ATG) is commonly used in allogeneic haematopoietic stem cell transplantation (HSCT). Little information is available, however, as to the optimal protocol for use and the side-effects occurring if ATG is administered in high daily doses (10-30 mg/kg). We report our experience with ATG Fresenius (ATG-F) in conditioning for allogeneic HSCT. During a period of 3 days, 47 patients received doses between 10 and 30 mg/kg either over 4 h preceded by 1-1.5 mg/kg prednisolone 30 min before the start of ATG-F (protocol A) or alternatively, over 12 h with 3-4 mg/kg prednisolone being administered before and 6 h after start of ATG (protocol B). During treatment with ATG-F, the side-effects observed included inflammation, disseminated intravascular coagulation, hyperdynamic circulation and renal dysfunction. Although these complications caused substantial morbidity, they were reversible within a few days. Side-effects were significantly more severe in patients treated according to protocol A than in those treated according to protocol B. As prolonged infusion of ATG-F does not reduce T cell clearance due to the long half-life of ATG-F, and since less cytokine release during conditioning might have beneficial long-term effects, we recommend administering ATG-F over 12 h preceded by high-dose steroid treatment.

Adolescent↗

PubMed2000

Gene-modified spontaneous Epstein-Barr virus-transformed lymphoblastoid cell lines as autologous cancer vaccines: mutated p21 ras oncogene as a model.

The transfer of genes coding for tumor antigens (Ags) into Ag-presenting cells is a promising strategy to develop cancer vaccines for patients not limited by major histocompatibility complex restriction. To test whether autologous lymphoblastoid cell lines (LCL) can be used as an alternative to dendritic cells for Ag presentation, we established LCL by spontaneous growth in cyclosporin-containing medium in vitro (SP-LCL). SP-LCL, which have an advantage over B95-8-transfected LCL in that they carry no risk of introducing a new infectious agent when used for vaccination, served as a permanent source for transfection with an episomal Epstein-Barr virus-based expression vector encoding the Ki-ras p21 oncogene carrying a point mutation at codon 12 (muRas) as a model tumor Ag. The ability of muRas-transfected SP-LCL (muRas-LCL) to induce immunoreactivity was determined in vitro. After electroporation with an optimized protocol, muRas-LCL expressed mutated ras peptides for a considerable period of time (at least 8 weeks) on the cell surface. The transfection procedure did not affect the expression of the costimulatory molecules B7.1, intercellular adhesion molecule-1, and leukocyte function associated Ag-3 by SP-LCL. muRas-LCL were able to induce muRas-specific cytotoxic T lymphocytes from three of three healthy donors and one of one patient with pancreatic carcinoma. Our results suggest that the gene transfer of muRas sequences to SP-LCL leads to an endogenous processing of this Ag in the major histocompatibility complex class I pathway and to functional presentation of antigenic peptides to CD8+ T lymphocytes. Autologous SP-LCL can serve as an unlimited renewable source for autologous cellular vaccines and appear to be good candidates as presenters for a wide range of human tumor Ags.

B7-1 Antigen↗

PubMed2002

T cell transduction and suicide with an enhanced mutant thymidine kinase.

Retroviral transfer of Herpes simplex virus thymidine kinase to T cells has been used to confer sensitivity to the antiviral agent ganciclovir. This has allowed therapeutic approaches to be developed in which T cells mediating graft-versus-host disease after bone marrow transplantation can be selectively eliminated by the administration of ganciclovir. Although the strategy has been shown to be generally successful in early clinical trials, there are concerns about possible resistance to ganciclovir and the risk of myelosuppressive side-effects at the doses required to induce T cell suicide. We have incorporated the enhanced mutant HSV-TKSR39 into retroviral vectors tailored to exhibit high levels of expression in T cells and have used protocols optimized for the transduction and selection of primary lymphocytes. We demonstrate that leukemic and primary T cells can be efficiently transduced and highly enriched under conditions that should be readily adaptable for clinical use. T cells carrying HSV-TKSR39 were inhibited by exposure to ganciclovir at concentrations an order of magnitude below those required for wild-type HSV-TK. The less toxic agent aciclovir also eliminated T cells transduced with HSV-TKSR39 (but not HSV-TK), underlining the increased therapeutic potential of the mutant suicide gene system in the bone marrow transplantation setting.

Acyclovir↗

PubMed2004

p-Iodophenol-enhanced luminol chemiluminescent assay applied to discrimination between acute lymphoblastic and minimally differentiated acute myeloid (FAB-M0) or acute megakaryoblastic (FAB-M7) leukemias.

INTRODUCTION: In this report, we propose the application of the p-iodophenol-enhanced luminol chemiluminescent technique to the determination of peroxidase (myeloperoxidase and/or platelet peroxidase) activity in blasts of minimally differentiated acute myeloblastic leukemia (AML-M0) and acute megakaryoblastic leukemia (AML-M7). METHODS: The frozen blast cells from 29 patients were thawed and submitted to the optimized protocol. RESULTS: All cases of AML-M7 and AML-M I exhibited integrated light emission greater than 73(10(2) mV x s), which was the arbitrary cutoff point set for the discrimination between AML and acute lymphoblastic leukemia (ALL) (mean + 3 x s.d. of ALL samples, n = 10). In addition, five out of seven cases of AML-MO showed results above the cutoff point. CONCLUSION: This highly sensitive enhanced chemiluminescent technique may be applied to discriminate between ALL and AML-M7 or AML-MI cases, and most AML-M0 cases. It is very simple, cheap and easy to perform compared to other procedures used to measure MPO activity in AML-leukemias including AML-M7 and AML-M0.

Bone Marrow Cells↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

PubMed2007

Kinetic properties of mutant deoxyguanosine kinase in a case of reversible hepatic mtDNA depletion.

DGUOK [dG (deoxyguanosine) kinase] is one of the two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA (mitochondrial DNA) replication. DGUOK is responsible for the initial rate-limiting phosphorylation of the purine deoxynucleosides, using a nucleoside triphosphate as phosphate donor. Mutations in the DGUOK gene are associated with the hepato-specific and hepatocerebral forms of MDS (mtDNA depletion syndrome). We identified two missense mutations (N46S and L266R) in the DGUOK gene of a previously reported child, now 10 years old, who presented with an unusual revertant phenotype of liver MDS. The kinetic properties of normal and mutant DGUOK were studied in mitochondrial preparations from cultured skin fibroblasts, using an optimized methodology. The N46S/L266R DGUOK showed 14 and 10% residual activity as compared with controls with dG and deoxyadenosine as phosphate acceptors respectively. Similar apparent negative co-operativity in the binding of the phosphate acceptors to the wild-type enzyme was found for the mutant. In contrast, abnormal bimodal kinetics were shown with ATP as the phosphate donor, suggesting an impairment of the ATP binding mode at the phosphate donor site. No kinetic behaviours were found for two other patients with splicing defects or premature stop codon. The present study represents the first characterization of the enzymatic kinetic properties of normal and mutant DGUOK in organello and our optimized protocol allowed us to demonstrate a residual activity in skin fibroblast mitochondria from a patient with a revertant phenotype of MDS. The residual DGUOK activity may play a crucial role in the phenotype reversal.

Cells, Cultured↗

PubMed1993

Reconstitution studies of amino acid transport system L in rat erythrocytes.

In many cell types, including human erythrocytes, membrane transport of hydrophobic amino acids such as leucine and phenylalanine is mediated primarily by Na(+)-independent system L. In this paper we demonstrate that erythrocytes from the rat have a 400-fold higher system L transport capacity than human erythrocytes. We have exploited this high transport activity to achieve the first successful reconstitution of an erythrocyte amino acid transporter into phospholipid vesicles. Rat erythrocyte membranes were depleted of extrinsic membrane proteins, solubilized in 50 mM n-octyl glucoside and reconstituted into egg-yolk phospholipid vesicles by a gel filtration freeze-thaw protocol. Optimal reconstitution of transport activity occurred at lipid/protein ratios of 25-35:1. At a lipid/protein ratio of 25:1, one-half of the total uptake of L-[14C]leucine (0.2 mM, 25 degrees C) was inhibited by 2 mM phloretin and thus judged to be carrier-mediated. This component of L-leucine uptake was inhibited by non-radioactive L-phenylalanine and L-leucine, and only to a very much weaker extent by glycine and L-alanine. Two other inhibitors of system L in intact cells, MK196 and PCMBS (p-chloromercuriphenylsulphonate), were also effective inhibitors of phloretin-sensitive L-leucine transport in reconstituted proteoliposomes. Phloretin-insensitive uptake of L-leucine in proteoliposomes occurred by simple diffusion across the lipid bilayer.

Amino Acids↗

PubMed1999

Therapeutic drug monitoring of antimetabolic cytotoxic drugs.

Therapeutic drug monitoring is not routinely used for cytotoxic agents. There are several reasons, but one major drawback is the lack of established therapeutic concentration ranges. Combination chemotherapy makes the establishment of therapeutic ranges for individual drugs difficult, the concentration-effect relationship for a single drug may not be the same as that when the drug is used in a drug combination. Pharmacokinetic optimization protocols for many classes of cytotoxic compounds exist in specialized centres, and some of these protocols are now part of large multicentre trials. Nonetheless, methotrexate is the only agent which is routinely monitored in most treatment centres. An additional factor, especially in antimetabolite therapy, is the existence of pharmacogenetic enzymes which play a major role in drug metabolism. Monitoring of therapy could include assay of phenotypic enzyme activities or genotype in addition to, or instead of, the more traditional measurement of parent drug or drug metabolites. The cytotoxic activities of mercaptopurine and fluorouracil are regulated by thiopurine methyltransferase (TPMT) and dihydropyrimidine dehydrogenase (DPD), respectively. Lack of TPMT functional activity produces life-threatening mercaptopurine myelotoxicity. Very low DPD activity reduces fluorouracil breakdown producing severe cytotoxicity. These pharmacogenetic enzymes can influence the bioavailability, pharmacokinetics, toxicity and efficacy of their substrate drugs.

Antimetabolites, Antineoplastic↗

PubMed2001

Therapeutic drug monitoring of cytotoxic drugs.

Therapeutic drug monitoring is not routinely used for cytotoxic agents. There are several reasons, but one major drawback is the lack of established therapeutic concentration ranges. Combination chemotherapy makes the establishment of therapeutic ranges for individual drugs difficult, the concentration-effect relationship for a single drug may not be the same as when that drug is used in a drug combination. Pharmacokinetic optimization protocols for many classes of cytotoxic compounds exist in specialized centres, and some of these protocols are now part of large multicentre trials. Nonetheless, methotrexate is the only agent which is routinely monitored in most treatment centres. An additional factor, especially in antimetabolite therapy, is the existence of pharmacogenetic enzymes which play a major role in drug metabolism. Monitoring of therapy could include assay of phenotypic enzyme activities or genotype in addition to, or instead, the more traditional measurement of parent drug or drug metabolites. The cytotoxic activities of mercaptopurine and fluorouracil are regulated by thiopurine methyltransferase (TPMT) and dihydropyrimidine dehydrogenase (DPD), respectively. Lack of TPMT functional activity produces life-threatening mercaptopurine myelotoxicity. Very low DPD activity reduces fluorouracil breakdown producing severe cytotoxicity. These pharmacogenetic enzymes can influence the bioavailability, pharmacokinetics, toxicity and efficacy of their substrate drugs.

Antimetabolites, Antineoplastic↗

PubMed2003

Composition of the additive solution affects red blood cell integrity after photodynamic treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment is a promising technique for pathogen inactivation of red blood cell concentrates. For protocol optimization, the influence of the composition of the storage solution on the integrity of phototreated red cells was studied. MATERIALS AND METHODS: Red blood cells were resuspended in the storage solutions SAG-M or AS-3 to a haematocrit (Hct) of 30%. After addition of the photosensitizer, 1,9-dimethylmethylene blue (DMMB) (25 microm), the suspensions were illuminated with red light, and potassium leakage and delayed haemolysis were determined. In some experiments, the cells were washed after illumination and resuspended in modified storage solutions. RESULTS: Illumination of red cells in the presence of DMMB resulted in an immediate, light-dose-dependent increase in potassium leakage. The illumination conditions used induced no detectable haemolysis immediately after photodynamic treatment. Potassium leakage was higher when the illumination was performed in AS-3. In contrast, delayed haemolysis, measured after overnight storage, was considerably lower when cells were stored in AS-3. This protection was mainly a result of the presence of citrate in AS-3. In addition, other impermeant solutes protected against haemolysis. CONCLUSIONS: The additive solution strongly influences the integrity of red cells after photodynamic treatment. Whereas the solution in which the cells are illuminated has a small effect on red cell integrity, the main influence of the additive solution is during post-treatment storage. Red cell integrity is best maintained when illumination is performed in SAG-M followed by storage in AS-3. The presence of non-permeant solutes, such as citrate, in the solution used for storage, prevents haemolysis of the phototreated, cation-permeable cells by counterbalancing the osmotic activity of haemoglobin.

Blood Preservation↗

PubMed2000

Nuclear medicine imaging in early vertebral osteomyelitis: still of clinical utility.

A case is presented of vertebral osteomyelitis in an elderly confused patient with poorly localizing signs. The lesion was not diagnosed on the initial MRI study of the spine due to poor targeting. The abnormality was detected on a bone scan the following day. This was confirmed with a gallium scan 3 days later, and also a repeat MRI study 11 days after the first MRI, using an optimized protocol over the region of interest established by the bone scan.

Aged↗