| 1. |
- Borg, Jörgen, et al.
(författare)
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Amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase.
- 2004
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 114:1-2, s. 21-30
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Tidskriftsartikel (refereegranskat)abstract
- Methods currently used for surface display on insect cells and budded baculovirus, all utilize the sequences from class I transmembrane proteins. This gives rise to some problems when handling unknown genes or cDNAs encoding full-length proteins. First, the stop codon from the cloned gene will be located upstream of the sequence for the transmembrane region. Second, the chance of getting the sequences encoding the signal peptide and the transmembrane region in frame with the cloned gene is small. To minimize these problems, we here present a method by which cDNAs or genes of interest can be cloned and fused to the codons for the signal peptide and transmembrane region of neuraminidase (NA), a class II transmembrane protein of the influenza virus. By placing both the signal peptide and transmembrane region at the amino-terminal, potential problems regarding stop codons are eliminated and errors in frame-shift minimized. To obtain proof of principle, the gene encoding enhanced green fluorescent protein, EGFP, was subcloned into a shuttle vector downstream of the neuraminidase sequence and the fusion product was then transferred to a baculovirus vector and transfected into insect cells (Sf9). Using this method, EGFP was found to be expressed on the surface of both infected cells and budded virus in an accessible manner.
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| 2. |
- Borgquist, Per, et al.
(författare)
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Simulation of the release from a multiparticulate system validated by single pellet and dose release experiments
- 2004
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Ingår i: Journal of Controlled Release. - 1873-4995. ; 97:3, s. 453-465
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Tidskriftsartikel (refereegranskat)abstract
- A previously described single-pellet release model has been simplified and modified to give predictions of the release from multiple-pellet systems, besides describing the release from single pellets. The simplified single-pellet model has been verified using single-pellet data and has been used to estimate three release-controlling parameters, namely the pellet core radius, the overall mass transfer coefficient, and the lag time. Single-pellet release experiments showed that the release from the individual film-coated drug cores resulted in a wide distribution of release profiles, a phenomenon not observed on the dose level. Therefore, the parameter estimations resulted in distributions of these parameter values. The core radius and the lag times compared well with the experimental data. The distributions were used as input data for the multiple pellet model, in order to predict the release profiles on the dose level, showing results consistent with the measured dose release. The dose-predictive ability of the model was demonstrated in simulations by studying the effect of a change in the size of the single subunits (of constant total dose), showing that smaller pellets give an increased release rate with less variation. The model for predicting dose-release profiles could be of great value in optimising the performance of an existing formulation, as well as in the development of a new control led-release pharmaceutical. (C) 2004 Elsevier B.V. All rights reserved.
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| 3. |
- Camire, Christopher, et al.
(författare)
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Material characterization and in vivo behavior of silicon substituted alpha-tricalcium phosphate cement.
- 2006
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Ingår i: Journal of Biomedical Materials Research. Part B - Applied Biomaterials. - : Wiley. - 1552-4981 .- 1552-4973. ; 76B:2, s. 424-431
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Tidskriftsartikel (refereegranskat)abstract
- The possibility and biological effects of substituting silicon in a-tricalcium phosphate (alpha-TCP) by way of solid-state reaction have been evaluated. a-TCP powders with varying substitution amounts (1 and 5 mol % Ca2SiO4) were synthesized by reacting mixtures of CaCO3, Ca(2)P(2)O7, and SiO2, at a rate of 4 degrees C(min)(-1) to 1100 degrees C, left to dwell for 2 h and then heated to 1325 degrees C at 4 degrees C(min)(-1) and left to dwell for a period of 4 h. The powders were then rapidly quenched in air. Si incorporation could be verified by X-ray diffraction analysis, indicating an increase of the lattice volume with increasing Si content from 4284.1(8) to 4334(1) angstrom(3) for pure alpha-TCP and alpha-Si5%TCP, respectively. The hydrolysis of milled alpha-SiTCP powders was monitored by isothermal calorimetry, and the compressive strength of set cements was tested. The results showed changes in speed and amount of heat released during reactivity tests and a decrease in mechanical strength (60, 50, and 5 MPa) with increasing Si content. In vitro bioactivity of the set cements after soaking in simulated body fluid for 4 weeks was also tested. The formation of a bonelike apatite layer on the surface of the set cements could be observed and was thickest for 1% Si (20 mu m). These results were in good agreement with the in vivo studies performed, which showed strong evidence that the cement containing 1% silicon doped alpha-TCP enhanced mesenchymal cell differentiation and increased osteoblast activity compared with alpha-TCP. (c) 2005 Wiley Periodicals, Inc.
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| 4. |
- Camire, Christopher, et al.
(författare)
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The effect of crystallinity on strength development of alpha-TCP bone substitutes.
- 2006
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Ingår i: Journal of Biomedical Materials Research. Part B - Applied Biomaterials. - : Wiley. - 1552-4981 .- 1552-4973. ; 79B:1, s. 159-165
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Tidskriftsartikel (refereegranskat)abstract
- Alpha phase tricalcium phosphates (alpha-TCP) were produced using a solid-state reaction method and milled for various periods of time. The resulting four materials were alpha-TCPs, ranging in crystalline content. Powders were exposed to X-ray diffraction for material identification as well as for use in crystallinity and purity calculations. Powder particle size was investigated using laser diffraction. Materials were mixed with 2.5% Na2HPO4 solution to initiate the hydration of alpha-TCP to calcium-deficient hydroxyapatite (CDHA). Isothermal calorimetry was performed to observe thermal response of the powders over a period of time. During the reaction process, at various time points up to 216 h, the material was compression tested to observe strength development. Materials proved to be predominantly alpha phase, while amorphous content determined by XRD varied. Reactivity, as measured by isothermal calorimetry, varied with crystallinity of the alpha-TCP powder. Speed of strength development did not change except for the most finely ground powder. In addition, crystal size of the CDHA was changed only in the product formed from the most highly ground material. It is proposed that increasing reactivity of alpha-TCP cements does not result in a corresponding increase in rate of strength development until there is sufficient supersaturation to produce significant crystal nucleation.
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| 5. |
- Fernandez, Celine, et al.
(författare)
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Hormone-sensitive lipase is necessary for normal mobilization of lipids during sub-maximal exercise.
- 2008
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Ingår i: American Journal of Physiology: Endocrinology and Metabolism. - : American Physiological Society. - 1522-1555 .- 0193-1849. ; 295, s. 179-186
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Tidskriftsartikel (refereegranskat)abstract
- For the working muscle there are a number of fuels available for oxidative metabolism, including glycogen, glucose and non-esterified fatty acids. Non-esterified fatty acids originate from lipolysis in white adipose tissue, from hydrolysis of VLDL-triglycerides or from hydrolysis of intramyocellular triglyceride stores. A key enzyme in the mobilization of fatty acids from intracellular lipid stores is hormone-sensitive lipase (HSL). The aim of the present study was to investigate the metabolic response of HSL-null mice challenged with exercise or fasting and to examine if other lipases are able to fully compensate for the lack of HSL. The results showed that HSL-null mice have reduced capacity to perform aerobic exercise. The liver glycogen stores were more rapidly depleted in HSL-null mice during treadmill exercise and HSL-null mice had reduced plasma concentrations of both glycerol and non-esterified fatty acids after exercise and fasting, respectively. The data support the hypothesis that in the absence of HSL mice are not able to respond to an exercise challenge with increased mobilization of the lipid stores. Consequently, the impact of the lipid sparing effect on liver glycogen will be reduced in the HSL-null mice, resulting in faster depletion of this energy source, contributing to the decreased endurance during sub-maximal exercise. Key words: Treadmill exercise, lipid metabolism, glycogen, skeletal muscle, liver.
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| 6. |
- Hansson, Ola, et al.
(författare)
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Transcriptome and proteome analysis of soleus muscle of hormone-sensitive lipase-null mice
- 2005
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Ingår i: Journal of Lipid Research. - 1539-7262. ; 46:12, s. 2614-2623
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Tidskriftsartikel (refereegranskat)abstract
- Hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization in adipocytes, has been demonstrated also in skeletal muscle. To gain further insight into the role and importance of HSL in skeletal muscle, a transcriptome analysis of soleus muscle of HSL-null mice was performed. A total of 161 transcripts were found to be differentially expressed. Increased mRNA levels of fructose-1,6-bisphosphatase, fructose-2,6-bisphosphatase, and phosphorylase kinase gamma 1A suggest a higher glycogen flux in soleus muscle of HSL-null mice. An observed increase in the utilization of glycogen stores supports this finding. Moreover, an increased amount of intramyocellular lipid droplets, observed by transmission electron microscopy, suggests decreased mobilization of lipid stores in HSL-null mice. To complement the transcriptome data, protein expression analysis was performed. Five spots were found to be differentially expressed: pyruvate dehydrogenase E1 alpha, creatine kinase (CK), ankyrin-repeat domain 2, glyceraldehyde-3-phosphate dehydrogenase, and one protein yet to be identified. The increased protein level of CK indicates creatine phosphate degradation to be of increased importance in HSL-null mice. The results of this study suggest that in the absence of HSL, a metabolic switch from reliance on lipid to carbohydrate energy substrates takes place, supporting an important role of HSL in soleus muscle lipid metabolism.
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| 7. |
- Holst, Martina, et al.
(författare)
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Subcellular distribution of spermidine/spermine N(1)-acetyltransferase
- 2008
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Ingår i: Cell Biology International. - : Wiley. - 1095-8355 .- 1065-6995. ; 32:1, s. 39-47
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Tidskriftsartikel (refereegranskat)abstract
- The subcellular distribution of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) was studied in L56Br-C1 cells treated with 10muM N(1),N(11)-diethylnorspermine (DENSPM) for 24h. Cells were fractioned into three subcellular fractions. A particulate fraction containing the mitochondria was denoted as the mitochondrial fraction. After DENSPM treatment, an increase in SSAT activity was mainly found in the mitochondrial fraction. Western blot analysis showed an increased level of the SSAT protein in the mitochondrial fraction compared to the cytosolic fraction. Immunofluorescence microscopy and immunogold labeling transmission electron microscopy also showed a mitochondrial association of SSAT. Transmission electron microscopy revealed that the endoplasmic reticulum was devoid of ribosomes in DENSPM-treated cells, in contrast to control cells that contained ample ribosomes. An increased SSAT activity in connection with the mitochondria may be part of the mechanism of DENSPM-induced apoptosis.
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| 8. |
- Jegou Saint-Jean, Simon, et al.
(författare)
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Study of the reactivity and in vitro bioactivity of Sr-substituted alpha-TCP cements
- 2005
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Ingår i: Journal of Materials Science: Materials in Medicine. - : Springer Science and Business Media LLC. - 1573-4838 .- 0957-4530. ; 16:11, s. 993-1001
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Tidskriftsartikel (refereegranskat)abstract
- In this study the effect of strontium substitution on the hydrolysis of alpha-tricalcium phosphate (alpha-TCP) toward the formation of calcium deficient hydroxyapatite (CDHA) was investigated. For that purpose substituted alpha-TCP powders with 1, 5 and 10 mol% Sr substitution for Ca were synthesized by reacting at 1500 degrees C stoichiometric amounts of CaCO3, SrCO3, and Ca2P2O7, followed by rapid quenching in air. XRD analysis of the powders revealed the presence of alpha-TCP (traces of beta-TCP) with enlarged unit cell volume at increased Sr contents, indicating the incorporation of Sr in the crystal structure. Strontium was also incorporated in the apatite phase as revealed by XRD analysis of the set cements. The hydrolysis of milled alpha-SrTCP powders and a pure alpha-TCP (control) was monitored by isothermal calorimetry and the compressive strength of set cements was tested. The results showed a decrease in the reactivity with increasing Sr content and similar final mechanical strength within the Sr series, though lower than the control. The in vitro bioactivity of the set cements after soaking in simulated body fluid for 4 weeks was also tested. The formation of a bone-like apatite layer on the surface of the set cements indicated a potential in vivo bioactivity. (C) 2005 Springer Science + Business Media, Inc.
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| 9. |
- Leal, Cecilia, et al.
(författare)
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Local and translational dynamics in DNA-lipid assemblies monitored by solid-state and diffusion NMR
- 2008
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1778:1, s. 214-228
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Tidskriftsartikel (refereegranskat)abstract
- The influence of electrostatic interactions on the dynamic properties of complexes containing DNA and mixtures of cationic- (DDA) and zwitterionic (DLPC) lipids are studied by means of NMR. The systems are arranged in lamellar membrane stacks intercalated by DNA molecules. This is confirmed by P-31-NMR, where a superposition of an axially symmetric powder pattern arising from the phospholipid membrane and an asymmetric tensor due to DNA can be fitted to the experimentally observed lineshape. The local mobility and order is assessed using two solid-state NMR techniques applicable to samples with natural isotopic abundance: WIdeline SEparation (WISE) and Separated Local Field (SLF) spectroscopy. Both experiments yield highly resolved C-13 spectra in the direct dimension. The indirect dimension contains information about molecular dynamics through the H-1 dipolar linewidth (WISE) or the H-1-C-13 dipolar coupling constant (SLF). The experiments suggest that DNA is static while it induces an increased disorder in the hydrocarbon chains as compared to the parent lipid case. DDA chain order is more affected than DLPC due to the attractive electrostatic interaction between DNA and the cationic lipid. Translational dynamics of the lipids and the water was measured with the Pulsed Field Gradient STimulated Echo (PFG STE) technique. The influence of lamellar domain size and the angular dependence of the diffusion coefficients and nuclear relaxation times on the results of the PFG STE experiments are discussed. The local water diffusion coefficient is reduced by a factor four from the value of bulk water, and increases as the DLPC content is increased. We observe two lipid components with an order of magnitude difference in diffusion coefficients in the DNA:DDA:DLPC precipitate and these are assigned to DLPC (fast) and DDA (slow). Cationic lipid (DDA) diffusion is decreasing a factor of 2 when DLPC is added to the pure DNA:DDA system, indicating DNA-induced lipid segregation within the bilayer and the transition from locally 2D to 1D diffusion of the DDA. The results show that DNA-lipid electrostatic interactions reduce the long-range lipid mobility but locally enhance the hydrocarbon chain dynamics by perturbing the preferred lipid packing.
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| 10. |
- Lindvall, Håkan, et al.
(författare)
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A novel hormone-sensitive lipase isoform expressed in pancreatic beta -cells.
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:5, s. 3828-3836
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Tidskriftsartikel (refereegranskat)abstract
- Hormone-sensitive lipase (HSL) is a key enzyme in fatty acid mobilization in many cell types. Two isoforms of HSL are known to date, namely HSLadi (84 kDa in rat) and HSLtes (130 kDa in rat). These are encoded by the same gene, with exons 1-9 encoding the parts that are common to both and an additional 5'-exon encoding the additional amino acids in HSLtes. HSL of various tissues, among these the islet of Langerhans, is larger than HSLadi, but not as large as HSLtes, indicating that there may be other 5'-coding exons. Here we describe the molecular basis for a novel 89-kDa HSL isoform that is expressed in -cells, adipocytes, adrenal glands, and ovaries in the rat and that is encoded by exons 1-9 and exon A, which is spliced to exon 1 and thereby introducing an upstream start codon. The additional 5'-base pairs encode a 43-amino acid peptide, which is highly positively charged. Conglomerates of HSL molecules are in close association with the secretory granules of the -cell, as determined by immunoelectron microscopy with antibodies targeting two separate regions of HSL. We have also determined that the human genomic sequence upstream of exon A has promoter activity in INS-1 cells as well as glucose sensing capability, mediating an increase in expression at high glucose concentration. The minimal promoter is present within 170 bp from the transcriptional start site and maximal glucose responsiveness is conferred by sequence within 850 bp from the transcriptional start site.
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