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PubMed2002

A study of the crystallisation of amorphous salbutamol sulphate using water vapour sorption and near infrared spectroscopy.

The crystallisation of amorphous salbutamol sulphate prepared by spray drying was monitored using a humidity controlled microbalance (Dynamic Vapour Sorption apparatus, Surface Measurement Systems) combined with a near-infrared probe. Amorphous salbutamol sulphate was prepared by spray drying from a solution in water. The particles were then analysed using scanning electron microscopy, thermogravimetric analysis, differential scanning calorimetry, powder X-ray diffraction, isothermal microcalorimetry and water vapour sorption analysis combined with near-infrared spectroscopy (NIR). Isothermal microcalorimetry and water vapour sorption combined with NIR spectroscopy were able to detect the transition from the amorphous to crystalline state. However while the isothermal microcalorimeter showed only a classic crystallisation exotherm when the material was exposed at 75% RH, the DVS-NIR results at the same humidity highlighted a more complex process. When exposed at 75% RH, the uptake of water was followed by crystallisation that was detected using NIR. The expulsion of water after crystallisation was very slow and at a constant rate whether the material was exposed to 75 or 0% RH. The NIR and DVS studies indicated that the material had crystallised very soon after exposure to high RH. The water that was expelled during crystallisation was not displaced from the particles and remained associated with the particles for many days. This study showed that the use of gravimetric analysis together with NIR spectroscopy provided valuable information on the dynamics of the crystallisation of salbutamol sulphate. The retention of water within recently crystallised salbutamol is potentially important to the behaviour of dosage forms containing the amorphous (or partially amorphous) form of this drug.

Albuterol↗
PubMedDate not supplied

[Isolation of a gamma heavy chain fragment from normal human serum].

Several components of catodic electrophoretic migration in serum and urine are present in normal individuals and in rabbit serum. There also exists in man and in some animals, serum fractions of low molecular weight. These types of serum components may be or may not be related with the IgG. In a previous study we have isolated two components in the slow catodic electrophoretic area of the normal human serum (NHS). One of them was identified as an IgG subclass and the other component presented a clear line of precipitation to gamma heavy chain specific immuno-serum. This latter component was found in the post gamma-globulin area crossing the IgG arc in the I.E. analysis. Its molecular weight was variable from 3700 to 9500. In this paper a differential analysis of the gamma fragment isolated for us, is made and its relationship with Fc subfragments of pepsin-digested IgG is studied. In order to obtain this comparative study, the electrophoresis, gel diffusion immunoelectrophoresis gel chromatography and analytic ultracentrifugation techniques are employed. The post-gammaglobulin fraction has been isolated from total normal human serum without previous manipulation, or with the gammaglobulin fraction precipitated with saturated ammonium sulphate, in Sephadex G-200 chromatography. These two fractions present similar immunelectrophoretical characteristics. The constant sedimentation is 0.90 S and the approximated molecular weight is 7000. Since the pepsin digestion of IgG produced Fc subfragments of low molecular weight, we have isolated and submitted this immunoglobulin to peptic digestion. The G-75 Sephadex filtration shows an isolated post-gamma-globulin of I.E. sedimentation constant and molecular weight whose characteristics are similar to the isolated serum post-gammaglobulin fraction. The antigenical analysis in I.D. shows a total identity between the pepsin digested post-gammaglobulin and the fragment obtained for us from the human serum to an anti-heavy gamma chain immuno-serum and to a rabbit serum anti-fraction produced for us. These events suggest that this isolation post-gamma-globulin fraction corresponds to a Fc subfragment of the IgG.

Animals↗
PubMed2004

Modeling first-hit functions of the t(12;21) TEL-AML1 translocation in mice.

The t(12;21) translocation, which generates the TEL-AML1 (ETV6-RUNX1) fusion gene, is the most common structural chromosome change in childhood cancer and is exclusively associated with the common B cell precursor subset of acute lymphoblastic leukemia (ALL). Evidence suggests that the translocation usually occurs in utero during fetal hemopoiesis and most probably constitutes an initiating or first-hit mutation that is necessary but insufficient for the development of overt, clinical leukemia. The mechanism by which TEL-AML1 contributes to this early stage of leukemogenesis is unknown. To address this question we have analyzed hemopoiesis in mice syngeneically transplanted with TEL-AML1-transduced bone marrow stem cells. TEL-AML1 expression was associated with an accumulation/expansion of primitive c-kit-positive multipotent progenitors and a modest increase in myeloid colony-forming cells. TEL-AML1 expression was, however, permissive for myeloid differentiation. Analysis of B lymphopoiesis revealed an increase in early, pro-B cells but a differentiation deficit beyond that stage, resulting in reduced B cell production in the marrow. TEL-AML1-positive B cell progenitors exhibited reduced expression of the surrogate light-chain component lambda5 and the IL-7 receptor, both of which may contribute to impedance of differentiation in vivo and account for their reduced in vitro clonogenicity in IL-7. A selective differentiation deficit of B lineage progenitors (i) is consistent with the phenotype of TEL-AML1-associated leukemia in children and (ii) provides a potential mechanism for the protracted preleukemic state that often precedes ALL. These results provide mechanistic insight into the role of the t(12;21) translocation in the initiation of common B cell precursor ALL.

Animals↗
PubMed2001

Glucagon-like peptide 1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting cells.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone capable of restoring normal glucose tolerance in aging glucose-intolerant Wistar rats. Whether the antidiabetic properties of GLP-1 are exclusively due to its insulin secretory activity remains to be determined. A GLP-1-dependent differentiation of pancreatic precursor cells into mature beta-cells has recently been proposed. The aim of this study was to investigate whether pancreatic ductal epithelial cells could be differentiated into insulin-secreting cells by exposing them to GLP-1. Rat (ARIP) and human (PANC-1) cell lines, both derived from the pancreatic ductal epithelium, were used to test this hypothesis. A major difference distinguishes these two cell lines: whereas ARIP cells spontaneously express the beta-cell differentiation factor islet duodenal homeobox-1 (IDX-1), PANC-1 cells are characteristically IDX-1 negative. GLP-1 induced the differentiation of ARIP cells into insulin-synthesizing cells, although it did not affect the phenotype of PANC-1 cells, as determined by fluorescence-activated cell sorting (FACS) analysis. Differentiation of ARIP cells by exposure to human GLP-1 occurs in a time- and dose-dependent manner, and this is associated with an increase in IDX-1 and insulin mRNA levels. Secretion of insulin was also induced in a parallel manner, and it was regulated by the concentration of glucose in the culture medium. Interestingly, PANC-1 cells, when stably transfected with human IDX-1, gained responsiveness to GLP-1 and were able to differentiate into beta-cells, as determined by FACS analysis, insulin gene expression, intracellular insulin content, and insulin accumulation in the culture medium. Finally, we demonstrated that the receptor for GLP-1 is constitutively expressed by ARIP and PANC-1 cells and that the mRNA level for this transcript was increased by cellular transfection with human IDX-1. In summary, our study provides evidence that GLP-1 is a differentiation factor for pancreatic ductal cells and that its effect requires the expression of IDX-1.

Actins↗
PubMed1995

The ovarian tumor protein isoforms of Drosophila melanogaster exhibit differences in function, expression, and localization.

The Drosophila melanogaster ovarian tumor (otu) gene, required for normal proliferation and differentiation of the female germ-line, encodes two cytoplasmic protein isoforms, 98 and 104 kDa. Mutants with defects in this gene are typically grouped into one of three phenotypic classes: quiescent (germ cells do not proliferate), oncogenic or tumorous (germ-line cells proliferate uncontrollably), and differentiated (germ-line cells initiate but do not complete differentiation). Analysis of transformants expressing only one of the otu isoforms showed that the 104-kDa isoform (otu-104) can rescue all classes of otu mutants, whereas only differentiated mutants are rescued to a significant extent by the 98-kDa isoform (otu-98). Western analysis of protein extracts prepared from ovaries of various developmental stages indicated that otu-104 predominates in predifferentiated stages, while otu-98 is prevalent in differentiated egg chambers. Immunolocalization experiments demonstrated that otu protein is present in the cytoplasm of oogonial stem cells that populate third instar larvae and in all germ-line-derived cells until late in oogenesis. In stage 10 egg chambers, otu protein shifts to the subcortical region of nurse cells. This type of analysis also showed that upon formation of a 16-cell syncytium otu-104, but not otu-98, preferentially accumulates in the developing oocyte cytoplasm. The otu mutant protein does not show this pattern of enhanced accumulation, nor does it occur in ovaries of egalitarian and Bicaudal-D mutants, which are defective in oocyte determination. Thus, these studies indicate that the 104-kDa isoform is required for normal proliferation of female germline cells and perhaps for oocyte differentiation. The 98-kDa isoform appears to be dispensable but can provide an otu function needed for the completion of oocyte maturation.

Animals↗
PubMed1993

The hydration of vimentin.

The hydration of vimentin isolated from the cortex of bovine lenses was studied by thermal analysis. Differential scanning calorimetry between -30, and 30 degrees C provided the freezable water content, and thermogravimetric analysis up to 105 degrees C yielded the total water content. The difference between the two gives the nonfreezable water content (bound water) as a function of concentration. The nonfreezable water content of the polymerized vimentin is higher than that of the monomeric vimentin. This behavior is exactly the opposite of that of actin. This difference is explained on the basis of different modes of supramolecular assembly in the two cytoskeletal bodies.

Animals↗
PubMed2006

A hybrid PHB-hydroxyapatite composite for biomedical application: production, in vitro and in vivo investigation.

Samples of a hybrid composite of polyhydroxybutyrate (PHB), a biodegradable polyester, and hydroxyapatite (HA), with different PHB/HA ratios, have been prepared using mechanical-physical method. Electron microscopy, X-ray structure analysis and differential thermal analysis have been used to investigate the structure and physicochemical properties of the composite, depending on the PHB/HA ratio. The properties of the surface of the HA-loaded composite are significantly different from those of the pure polymer. As the HA percentage in the composite increases, free interface energy, the cohesive force, i.e., the strength of the adhesive bond between the composite surface and the water phase, and surface wettability increase. The HA percentage of the composite does not influence its melting temperature, but affects the temperature for the onset of decomposition: as the HA content increases from 0 to 10% (w/w), Td decreases from 260 degrees C to 225 degrees C. The degree of crystallinity of PHB/HA increases from 77% to 89% with an increase in the HA fraction from 10% to 50%. Functional properties of the composites have been investigated in vitro and in vivo. The best parameters of growth and differentiation of murine marrow osteoblasts are registered on PHB/HA samples containing 10% and 20% HA. In ectopic bone formation assay it has been proven that the hybrid PHB/HA composites can function as scaffolds and that bone tissue develops on their surface and in pores.

Animals↗
PubMed2004

DNA microarray normalization methods can remove bias from differential protein expression analysis of 2D difference gel electrophoresis results.

MOTIVATION: Two-dimensional Difference Gel Electrophoresis (DIGE) measures expression differences for thousands of proteins in parallel. In contrast to DNA microarray analysis, however, there have been few systematic studies on the validity of differential protein expression analysis, and the effects of normalization methods have not yet been investigated. To address this need, we assessed a series of same-same comparisons, evaluating how random experimental variance influenced differential expression analysis. RESULTS: The strong fluctuations observed were reflected in large discrepancies between the distributions of the spot intensities for different gels. Correct normalization for pooling of multiple gels for analysis is, therefore, essential. We show that both dye-specific background levels and the differences in scale of the spot intensity distributions must be accounted for. A variance stabilizing transform that had been developed for DNA microarray analysis combined with a robust Z-score allowed the determination of gel-independent signal thresholds based on the empirical distributions from same-same comparisons. In contrast, similar thresholds holding up to cross-validation could not be proposed for data normalized using methods established in the field of proteomics. AVAILABILITY: Software is available on request from the authors. SUPPLEMENTARY INFORMATION: There is supplementary material available online at http://www.flychip.org.uk/kreil/pub/2dgels/

Algorithms↗
PubMed2005

New hybrid polymeric liquid chromatography chiral stationary phase prepared by surface-initiated polymerization.

A new hybrid organic/inorganic HPLC chiral stationary phase (CSP1) has been synthesized by the grafting from (g-from) radical polymerization of an enantiopure diacryloyl derivative of trans-1,2-diaminocyclohexane in the presence of mesoporous, azo-activated silica particles. The new chiral stationary phase has been fully characterized by elemental analysis, differential scanning calorimetry, diffuse reflectance infrared spectroscopy, scanning electron microscopy, inverse size exclusion chromatography and Van Deemter analysis. CSP1 shows improved chromatographic performances compared to its analog CSP2 synthesized by the alternative grafting to (g-to) approach in which the azo initiator is kept in solution. CSP1 can successfully resolve several chemically diverse chiral compounds, using both organic and water-based eluents (normal phase, polar organic, etc.).

Calorimetry, Differential Scanning↗
PubMed1982

Sex determination in Drosophila melanogaster: analysis of transformer-2, a sex-transforming locus.

The transformer-2 (tra-2) locus is one of a set of regulatory loci that control sex determination in Drosophila melanogaster. Temperature-shift experiments with temperature-sensitive tra-2 mutants demonstrate that within single cell lineages tra-2+ function is required at several times, and probably continuously, during development for the occurrence of a series of determinative decisions necessary for female sexual differentiation. Analysis of the effects of tra-2 in the genital disc demonstrates that the tra-2+ function is necessary in females both to prevent male sexual differentiation and to permit female differentiation. These and other results support the model that the tra-2+ and tra+ loci act to control the expression of the bifunctional doublesex (dsx) locus.

Animals↗
PubMed1983

Use of a microcomputer in the intensive care unit.

A microcomputer was programmed by ICU personnel to serve as a ready-reference manual data organizer, and source of continuing education. All members of the ICU staff can use the system at any time and no prior training in computer technology is required. Ten programs dealing with common clinical situations are in use, including arterial blood gas analysis, electrolyte analysis, differential diagnosis of shock, adverse drug interactions, inotrope calculations and infusion rates, ventilator management and trouble-shooting, management of intravenous hyperalimentation, derived cardiovascular variables, and coronary risk factor analysis and management. Medication errors have almost been eliminated with use of the computer. Total cost of the system is less than $5000.

Asthma↗
PubMed1988

Application of bromodeoxyuridine incorporation measurements to the determination of cell distribution within the S phase of the cell cycle.

Flow cytofluorimetric measurement of incorporated bromodeoxyuridine, using a double-stained cell population, allows the determination of the distribution of cells along the cell cycle. We have developed a simple computer program for the direct treatment of 64 x 64 channel histograms. This analysis appears to provide interesting data about the distribution of cells in the various phases of the cell cycle, namely the S phase. Two examples have been chosen to illustrate possible fields for the application of such a program. Comparison of two cell lines such as friend murine erythroleukemia cells (MELC) and fibroblasts FR3T3 cells has shown that this analysis can be used for cell-cycle characterization of a given cell line. The program also allows the differential analysis of cell distribution along the cell cycle as a function of a given parameter. This possibility has been applied to study the variation of cell-cycle parameters as a function of the time of induced differentiation of MELC and reveals changes in the distribution of the cells along the various phases of the cell cycle, namely in the S phase.

Animals↗
PubMed2006

[Enhancement of all-trans retinoic acid-induced differentiation by bufalin in primary culture of acute promyelocytic leukemia cells].

OBJECTIVE: To investigate the effect of bufalin combined with all-trans retinoic acid-induced (ATRA) differentiation of acute promyelocytic leukemia (APL) cells in primary culture. METHODS: Fresh leukemia cells were obtained from heparinized bone marrow aspirations of 12 newly diagnosed APL patients. Cell viability was determined by trypan blue dye exclusion. Apoptosis of APL cell was assessed by morphological analysis. Differentiation of APL cell was also assessed by morphological analysis. Nitro blue tetrazolium (NBT) reduction test and expression of the granulocyte/macrophage-specific antigen CD(11)b was carried out with flow cytometric assay. RESULTS: Bufalin combined with ATRA can induce differentiation of APL cells towards mature stages, NBT reduction was increased 15% - 52% and CD(11)b expression was also increased 16% - 69% in combination of bufalin and ATRA as compared with that of ATRA alone, while the concentration of ATRA needed in the combination group was reduced to 30% and the time of differentiation was reduced from 7 days to 4 days. CONCLUSION: The combination of ATRA with bufalin can significantly enhance the differentiation of acute promyelocytic leukemia cells in primary culture by ATRA.

Antineoplastic Agents↗
PubMed2003

Protein arrays: the current state-of-the-art.

The completion of projects for sequencing whole genomes such as those of human, Saccaromyces cerevisiae and Caenorhabditis elegans has led to a rapid increase in the availability of genetic information. The technology by which such information is acquired is having a major impact on the way we think about analysing the RNA and protein products of the gene transcription. Transcriptome analysis has, via microarray technology, managed to achieve a near genomic scale quantitative analysis of mRNA levels. Several other technologies such as quantitative reverse transcriptase polymerase chain reaction and representational differential analysis are also available for analysing gene products. Traditionally, protein analysis has been performed by assaying one particular protein at a time, with very little parallel analysis. As protein function is a direct consequence of the protein product of the gene and as mRNA levels do not always correlate well with protein, it is desirable to analyse the entire protein complement of a cell etc. on a similar scale to mRNA. Despite the clear interest in analysing the expression, structure and function of proteins at a genomic scale, they have proven less amenable to such generic, high-throughput approaches. This review highlights the current thinking in the area of proteome analysis and considers the potential for future technology development in the area of protein arrays.

Animals↗
PubMed1997

Expression of three UDP-N-acetyl-alpha-D-galactosamine:polypeptide GalNAc N-acetylgalactosaminyltransferases in adenocarcinoma cell lines.

The levels of mRNA expression of three UDP-N-acetyl-alpha-D-galactosamine:polypeptide GalNAc N-acetylgalactosaminyltransferases (GalNAc-transferases) were quantified for human adenocarcinoma cell lines from pancreas, colon, stomach, and breast. Two of the GalNAc-transferases, GalNAc-T1 and GalNAc-T2, were expressed constitutively and at low levels in most or all cell lines examined. A third GalNAc-transferase, GalNAc-T3, was differentially expressed. Well-differentiated adenocarcinoma cell lines expressed high levels and moderately differentiated cell lines expressed lower levels of GalNAc-T3. Cell lines classified as poorly differentiated failed to express GalNAc-T3 mRNA at levels that could be detected by Northern blot analysis. Differential expression of the GalNAc-T3 protein was confirmed in these cell lines by Western blotting. We propose that glycosylation in tumor cell lines may be regulated in part by differential expression of GalNAc-transferases, and we suggest that GalNAc-T3 gene expression may be a molecular indicator of differentiated adenocarcinoma.

Actins↗
PubMed1998

Identification and cloning of differentially expressed genes by long-distance differential display.

Differential mRNA display (DD-PCR) amplifies short cDNAs (average size 100-350 bp), representing mainly the 3' untranslated regions (3' UTR) of transcripts. Sequencing of these cDNAs is predominantly uninformative for prediction of function and selection of clones for further analysis. Differential display of longer amplicons (0.5-2.0 kb) could enable isolation of cDNAs that encompass both 3' UTR and at least part of the 3' end of the coding region. The coding sequence information could facilitate selection of candidate clones for further analysis without the necessity of screening cDNA libraries. By combining DD-PCR protocols with long-distance PCR and using hot-start PCR with rTth DNA polymerase we have successfully amplified and comparatively displayed cDNAs ranging in size from 150 bp to 2 kb. Long-distance DD-PCR (LDD-PCR) has generated highly reproducible primer-specific patterns of cDNA fragments, as well as reproducible duplicate fingerprints, obtained from different RNA and cDNA samples. Sequencing and expression analyses of LDD-PCR clones have shown that LDD-PCR (a) enables nonredundant clone sampling, (b) generates many clones that encompass part of the coding region, and (c) samples both abundant and rare transcripts, approximately 60% of which are differentially expressed as confirmed by Northern analysis. Coupled with high-throughput cDNA sequencing and multiplex hybridization of cDNA microarrays for confirmation of differential expression, LDD-PCR could prove to be useful for simultaneous scanning of transcripts from multiple cDNA samples and faster selection of differentially expressed transcripts of interest.

Animals↗
PubMed2002

Molecular analysis of expansion, differentiation, and growth factor treatment of human chondrocytes identifies differentiation markers and growth-related genes.

This study is intended to optimise expansion and differentiation of cultured human chondrocytes by growth factor application and to identify molecular markers to monitor their differentiation state. We dissected the molecular consequences of matrix release, monolayer, and 3D-alginate culture, growth factor optimised expansion, and re-differentiation protocols by gene expression analysis. Among 19 common cartilage molecules assessed by cDNA array, six proved best to monitor differentiation. Instant down-regulation at release of cells from the matrix was strongest for COL 2A1, fibromodulin, and PRELP while LUM, CHI3L1, and CHI3L2 were expansion-related. Both gene sets reflected the physiologic effects of the most potent growth-inducing (PDGF-BB) and proteoglycan-inducing (BMP-4) factors. Only CRTAC1 expression correlated with 2D/3D switches while the molecular phenotype of native chondrocytes was not restored. The markers and optimised protocols we suggest can help to improve cell therapy of cartilage defects and chondrocyte differentiation from stem cell sources.

Becaplermin↗
PubMed1984

The influence of fibroblast-like cells derived from canine fetal hematopoietic tissues on the regulation of lymphohematopoiesis.

Media conditioned by fibroblast-like cells derived from organs active in fetal lymphohematopoiesis were studied for their effects on adult granulocyte/macrophage colony-forming units (CFU-GM). Fibroblasts from fetal liver produced a factor stimulatory for CFU-GM, whereas fibroblasts from fetal marrow produced a factor inhibitory for CFU-GM which was not completely relieved by adding indomethacin to the assay. Our studies indicated that neither fetal marrow nor fetal liver produced factors affecting lymphocyte colony-forming units (CFU-L). Cell-cell interactions between fibroblast-like cells derived from fetal liver or marrow and normal adult CFU-GM were also studied. We observed that fibroblasts derived from both fetal and adult marrow inhibited colony formation, whereas inhibition in the presence of fetal liver fibroblasts was minimal. Loss of inhibitory activity by a liver fibroblast cell line over repeated passages was seen. Differential analysis of colonies formed above an adherent layer of fetal marrow fibroblasts suggested that these fibroblasts suppress myeloid/macrophage differentiation to a far greater degree than did adult marrow fibroblasts. A role in the regulation of fetal lymphohematopoiesis may be played by stromal fibroblasts.

Animals↗