| 1. |
- Ahuja, Sat pal, et al.
(författare)
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Glutathione S-transferase µ(GST) modifies activities of proteases and levels of cystatin C secreted by mouse retinal explants
- 2004
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Ingår i: Investigative Ophthalmology & Visual Science. - 1552-5783. ; 45, s. 352-352
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Konferensbidrag (refereegranskat)abstract
- Purpose: In one form of human autosomal recessive retinitis pigmentosa and in retinal degeneration (rd1) mouse, mutation occurs in the genes encoding ß subunit of rod photoreceptor cGMP phosphodiesterase. Therefore, rd1 mutant mouse is an appropriate model for human inherited retinal degeneration studies. Retinal explants are successfully cultured in serum free chemically defined R16 medium to evaluate effects of various rescue factors and retinal conditioned medium (RCM) for secreted molecules like proteases and their inhibitors. Cysteine protease inhibitor cystatin C has recently been identified in rodent neuroretina and RPE. RCM of explants treated with GST were analyzed for proteases and cystatin C to explain, in part, mode of action of GST in protection of degenerating retina. Methods: Postnatal day 2 (PN2) and PN7 control (wt) and rd1 were cultured with (10 ng / ml GST) and without GST in R16 medium, respectively, for 26 and 21 days in vitro (div). Retinal extracts (RE) and RCM were analyzed by fluorometry using casein green fluorescent labeled with BODIPY–FL (Molecular Probes) for total proteases; Z–Phe–Arg–NMec or Z–Arg–Arg–NMec for cysteine proteases and by ELISA for cystatin C, respectively, for levels and secretion of proteases and cystatin C. The protein content of RE was measured. Results: Protein content (µg) of RE from wt and rd1 retinal extracts respectively increased and decreased with age. Cystatin C (ng/ml RCM) content in wt and rd1 RE increased with age (was always higher in wt) up to PN14 and then decreased but was higher than that at PN2. Progressive secretion of cystatin C by PN2 explants was lower than that by PN7 explants; and that by rd1 PN2 and PN7 explants was initially lower up to in vitro age of PN19 and subsequently it was higher than that by wt explants. Secretion of total cystatin C by PN2 and PN7 wt and rd1 explants was similar and was increased by GST. During initial stage of culture total protease activity ({Delta} F / 100 µl RCM) in RCM of rd1 PN2 and PN7 explants was higher and was decreased in GST treated explants. Conclusions: Cystatin C content and secretion by wt RE is always higher and that of proteases is lower than that of rd1. Treatment with GST increases content of cystatin C and consequently decreases that of proteases especially cysteine proteases.
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| 2. |
- deWeert, Michael J., et al.
(författare)
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Analysis of spatial variability in hyperspectral imagery of the uterine cervix in vivo
- 2003
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Ingår i: Proceedings of SPIE. - : SPIE. ; 4959, s. 67-76
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Tidskriftsartikel (refereegranskat)abstract
- The use of fluorescence and reflectance spectroscopy in the analysis of cervical histopathology is a growing field of research. The majority of this research is performed with point-like probes. Typically, clinicians select probe sites visually, collecting a handful of spectral samples. An exception to this methodology is the Hyperspectral Diagnostic Imaging (HSDI®) instrument developed by Science and Technology International. This non-invasive device collects contiguous hyperspectral images across the entire cervical portio. The high spatial and spectral resolution of the HSDI instruments make them uniquely well suited for addressing the issues of coupled spatial and spectral variability of tissues in vivo. Analysis of HSDI data indicates that tissue spectra vary from point to point, even within histopathologically homogeneous regions. This spectral variability exhibits both random and patterned components, implying that point monitoring may be susceptible to significant sources of noise and clutter inherent in the tissue. We have analyzed HSDI images from clinical CIN (cervical intraepithelial neoplasia) patients to quantify the spatial variability of fluorescence and reflectance spectra. This analysis shows the spatial structure of images to be fractal in nature, in both intensity and spectrum. These fractal tissue textures will limit the performance of any point-monitoring technology.
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| 3. |
- El-Sukkari, D, et al.
(författare)
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The protease inhibitor cystatin C is differentially expressed among dendritic cell populations, but does not control antigen presentation
- 2003
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Ingår i: Journal of Immunology. - 1550-6606. ; 171:10, s. 5003-5011
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Tidskriftsartikel (refereegranskat)abstract
- Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class H (MHC H) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC H chaperone E, and hence in the formation of MHC H-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8(+) DC, but not CD4(+) or CD4(-)CD8(-) DC, synthesize CyC, which accumulates in MHC II(+)Lamp(+) compartments. However, II processing and MHC H peptide loading proceeded similarly in all three DC populations. We then analyzed MHC H localization and Ag presentation in CD8(+) DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.
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| 4. |
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| 5. |
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| 6. |
- Håkansson, Anders, et al.
(författare)
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Forskningstræning for alle læger - også uden for universitetssygehusene
- 2003
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Ingår i: Ugeskrift for Læger. - 0041-5782. ; 165:36, s. 3423-3423
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: General practitioners are often interested in doing research but are hampered by lack of time and research training. Interpreting the results of others can also be difficult. For this reason a course in basic research methods for GPs was started in Sweden in 1989. It was originally aimed at GPs but was later extended to hospital physicians as well. The course is given regionally and at present is held in six different locations in southern Sweden as well as in Hillerod, Denmark. The aim of this study is to evaluate the course as part of a research project to recommend changes to the courses in accordance with the course evaluations. MATERIALS AND METHODS: The course consists of theory (lectures/seminars six hours a month) and practice (project work) over a period of 18 months. Questionnaires were mailed to the 112 physicians who, starting in 1997 and 1999, completed the course. Eighty-five percent responded to the questionnaire, which asked if the course goals were relevant and if they thought they had achieved them. RESULTS: The most frequent reasons given for attending the course were a desire to learn how to read scientific articles critically and how to carry out one's own research projects. Two thirds of the participants thought that the theoretical lectures and project work had supplemented each other well. Most of the participants thought that the goals were very relevant but fewer--between 57% and 77%--felt those goals had been achieved. DISCUSSION: The course provides important empirical information with regard to future specialist training in Denmark. The plan is for all physicians to have research training--amounting to a total of 60 days--with lectures in research methodology and tutored research projects. The Danish College of General Practitioners has planned research training in accordance with the experience from these courses.
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| 7. |
- Håkansson, Katarina
(författare)
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Evaluation of MRI as a diagnostic method in biliary tract conditions
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The diagnostic value of modern fast pulse sequences and breath-hold technique was evaluated in a prospective study of patients with suspected biliary diseases. The cost-effectiveness of MRI compared to ultrasound was calculated. Characteristics of acute cholecystitis and cholangitis were evaluated, the appearance of bile in MRCP with reference to its chemical and physical properties was studied, as well as MRI appearance after cholecystectomy with and without postoperative abdominal complications. The sensitivity and specificity to diagnose acute cholecystitis was 88% and 89% for MRI and 65% and 89% for US. The sensitivity of US, MRI and ERCP for correct diagnosis in jaundiced or not jaundiced patients was 53, 93 and 89% and 50, 100, and 70%, respectively. MRI is superior to US giving correct diagnosis in patients with right upper quadrant abdominal pain or to diagnose biliary tract conditions. Screening with US and supplemental MRI in non-diagnostic cases was estimated to cost 80% of the total amount compared to examining with MRI only. Relaxation rates 1/T1 and 1/T2 of gallbladder bile in vitro showed large inter-individual variation but a consistent linear relationship was found. The relaxation rates increased with increasing amounts of substances in the bile. No single chemical parameter showed dominating influence on relaxation rates. Occasionally bile shows very high relaxation rates leading to unsuccessful MRCP. Early MR findings following cholecystectomy consist normally of only subtle changes, mainly in the gallbladder fossa. A postoperative surgical complication is unlikely if MRI fails to show a fluid collection. Acute cholangitis has multiple findings on MRI, biliary ductal dilatation was the most consistent finding and inflammatory changes were observed in 50% of patients. MRI has the potential to confirm the diagnosis acute cholangitis and exclude alternative diagnosis especially in older patients where the clinical symptoms may be vague. MRI is a reliable method providing high quality images performed with a minimum of inconvenience for the patient. In the future the non-invasive MRI will play a greater role as the primary imaging technique to provide maximum clinical utility when the MRI system will be even more easy to manage, faster, more cost-efficient and, compared with CT, MRI has a significant advantage since no ionizing radiation is involved.
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| 8. |
- Håkansson, Mikael, 1957, et al.
(författare)
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Copper(I) complexes with conjugated dienes
- 2000
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Ingår i: Journal of Organometallic Chemistry. - 0022-328X. ; 602:1-2, s. 5-14
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Tidskriftsartikel (refereegranskat)abstract
- Three copper(I) complexes containing conjugated dienes and one containing the acetylenic analogue of isoprene, 2-methyl-1-butene-3-yne (isopropenylacetylene), have been prepared and characterised by means of crystal-structure determination. Direct reaction between isoprene (2-methylbutadiene) and copper(I) trifluoromethylsulfonate, using triphenylphosphine as a stabilising ligand, results in [Cu-2(PPh3)(2)(mu-(H2C=CHC(CH3)=CH2))(O3SCF3)(2)] (1), whereas reaction between copper(I) chloride and isopropenylacetylene, dimethylbutadiene, and trans-1,3-pentadiene yields the labile compounds [Cu2Cl2 (mu-(H2C=CHC(CH3)=CH))] (2), [Cu2Cl2 (mu-(H2C=C(CH3)C(CH3)-CH2))] (3) and [Cu2Cl2(mu-(trans-H2C=CHCH=CH(CH3))] (4), respectively. All four compounds are polymeric. Thus, the organic ligands bridge two copper(I) centres via the alkene or alkyne bonds, the conjugated dienes all assuming the s-trans conformation. Further bridging is effected by the trifluoromethylsulfonate ligands in 1 and by the chloride ligands in 2, 3 and 4. In 1, copper(I) has distorted tetrahedral geometry, whereas in 2, 3 and 4, copper(I) exhibits tetrahedral or trigonal pyramidal geometry with the C=C/C=C linkage in the trigonal plane and an apical Cu-Cl bond. The nearly planar carbon skeleton of isoprene in 1 bonds to two copper(I) atoms from opposite faces of the diene. The Cu-C distances range from 2.085(6) to 2.220(6) Angstrom [C(CH3)] and the C=C bond lengths are 1.360(8) and 1.353(8) Angstrom. Trifluoromethylsulfonate ligands bridge adjacent [Cu-2(PPh3)(2)(mu-(H2C=CHC(CH3)-CH2))(O3SCF3)(2)] units leading to the formation of chains, containing internal chair-shaped Cu2S2O4 rings alternating with isoprene ligands, with peripheral triphenylphosphine ligands. The carbon skeleton of the isopropenylacetylene ligand in 2 and those of dimethylbutadiene in 3 and of trans-1,3-pentadiene in 4 are also approximately planar, but, unlike the situation in 1, in compounds 2-4, two copper(I) atoms are coordinated by C=C/C=C from the same face of the ligand. In 2, Cu-C distances range from 2.005(11) to 2.158(9) Angstrom [C(CH3)] and the C=C and C=C bond lengths are 1.373(13) and 1.200(14) Angstrom, respectively. In 3, Cu-C distances range from 2.06(1) to 2.17(1) Angstrom [C(CH3)] and both C=C bonds are 1.35(1) Angstrom. Dimeric [Cu2Cl2(mu-(H2C=CHC(CH3)=CH))](2) and [Cu2Cl2(mu-(H2C=C(CH3)C(CH3)=(CH2))] units are linked by long Cu-Cl bonds leading to the formation of chains with peripheral isopropenylacetylene (2) and dimethylbutadiene (3) ligands. The crystal structure of 4 could be determined only with low precision, but can be described in terms of copper(I) chloride layers with peripheral trans-1,3-pentadiene ligands. Shifts in the infrared absorptions on coordination of the conjugated dienes (including butadiene and cis-1,3-pentadiene) to copper(I) are discussed in the light of the crystal structures of 2-4.
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| 9. |
- Wasselius, Johan, et al.
(författare)
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Cystatin C in the anterior segment of rat and mouse eyes.
- 2004
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Ingår i: Acta Ophthalmologica Scandinavica. - : Wiley. - 1395-3907 .- 1600-0420. ; 82:1, s. 68-75
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Cystatin C is a mammalian cysteine protease inhibitor. This study describes the localization of cystatin C in the anterior segment of normal rat and mouse eyes. Cysteine proteases play an important role in protein degradation (e.g. of photoreceptor outer segments in the retinal pigment epithelium) and the balance between these proteases and their specific inhibitors is therefore of great interest. Methods: Cells containing cystatin C were identified by immunohistochemistry and quantified by ELISA. Messenger RNA levels were analysed by quantitative real-time polymerase chain reaction. Results: Cystatin C is present at biologically significant levels in the corneal epithelium, endothelium and stromal keratinocytes, lens epithelium, epithelial cells in the ciliary processes, aqueous humour and iris stromal cells. In the rat anterior segment, the highest cystatin C concentrations were found in the ciliary epithelium. Conclusions: Cystatin C is present in several cell types and is probably locally produced. The inhibitor is likely to be an important regulator of cysteine proteases in the retinal pigment epithelium, ciliary epithelium, aqueous humour, lens epithelium and in the corneal endothelium and epithelium.
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| 10. |
- Wasselius, Johan, et al.
(författare)
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Identification and localization of retinal cystatin C
- 2001
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Ingår i: Investigative Ophthalmology & Visual Science. - 1552-5783. ; 42:8, s. 1901-1906
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE. Cystatin C is a mammalian cysteine protease inhibitor, synthesized in various amounts by many kinds of cells and appearing in most body fluids. There are reports that it may be synthesized in the mammalian retina and that a cysteine protease inhibitor may influence the degradation of photoreceptor outer segment proteins. In the current study cystatin C was identified, quantitated, and localized in mouse, rat, and human retinas. METHODS. Enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), DNA sequencing, Western blot analysis, and immunohistochemistry have been used on mouse, rat, and human retinas (pigment epithelium included). RESULTS. Cystatin C is present in high concentrations in the normal adult rat retina, as it is throughout its postnatal development. Its concentration increases to a peak at the time when rat pups open their eyes and then remains at a high level. It is mainly localized to the pigment epithelium, but also to some few neurons of varying types in the inner retina. Cystatin C is similarly expressed in normal mouse and human retinas. CONCLUSIONS. Cystatin C was identified and the localization described in the retinas of rat, mouse, and human using several techniques. Cystatin C is known to efficiently inactivate certain cysteine proteases. One of them, cathepsin S, is present in the retinal pigment epithelium and affects the proteolytic processing by cathepsin D of diurnally shed photoreceptor outer segments. Hypothetically, it appears possible that retinal cystatin C, given its localization to the pigment epithelium and its ability to inhibit cathepsin S, could be involved in the regulation of photoreceptor degradation.
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