| 1. |
- Beckman-Sundh, Ulla, et al.
(författare)
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A screening method for phosphohistidine phosphatase 1 activity
- 2011
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Ingår i: Upsala Journal of Medical Sciences. - : Uppsala Medical Society. - 0300-9734 .- 2000-1967. ; 116:3, s. 161-168
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Tidskriftsartikel (refereegranskat)abstract
- Introduction. Research in the field of protein-bound phosphohistidine phosphorylation has been hampered by the difficulties in analysis and detection of phosphohistidine. Therefore a screening method was developed primarily for the analysis of phosphohistidine phosphatase 1 (PHPT1) activity. Methods. A highly positively charged substrate, Ac-Val-Arg-Leu-Lys-His-Arg-Lys-Leu-Arg-pNA, containing the peptide surrounding the phosphorylated histidine in ion channel KCa3.1 was chemically phosphorylated using phosphoramidate. Excess phosphoramidate was removed by anion exchange chromatography using a micro spin column. After incubation of the eluate with PHPT1, the removed phosphate was bound on a consecutive anion exchange spin column. The eluate was assayed in a micro plate format for remaining phosphate in the substrate Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA. Histone H4, also highly positive in charge, was subjected to the same procedure to explore the possibility to use other substrates to PHPT1 in this assay format. Results. It was found that Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA and phosphohistone H4 were dephosphorylated by PHPT1. The apparent K(m) for Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA was in the order of 10 mu M. Using this method, phosphohistidine phosphatase activity was detected in mouse liver cell sap with Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA as substrate. Discussion. The described method for determination of PHPT1 activity is comparably much easier and faster than presently used methods for detection of phosphohistidine phosphatase activity. It is also sensitive, since the lower activity limit was 5 pmol phosphate released per min. It has the potential to be used both for more rapid screening for inhibitors and activators to phosphohistidine phosphatases and for screening of histidine kinases.
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| 2. |
- Ek, Pia, et al.
(författare)
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Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase
- 2002
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 269, s. 5016-5023
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Tidskriftsartikel (refereegranskat)abstract
- Protein histidine phosphorylation in eukaryotes has beensparsely studied compared to protein serine/threonine andtyrosine phosphorylation. In an attempt to rectify this byprobing porcine liver cytosol with the phosphohistidinecontainingpeptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide(phosphopeptide I), we observed a phosphataseactivity that was insensitive towards okadaic acid andEDTA. This suggested the existence of a phosphohistidinephosphatase different from protein phosphatase 1, 2Aand 2C. A 1000-fold purification to apparent homogeneitygave a 14-kDa phosphatase with a specific activity of 3lmolÆmin)1Æmg)1 at pH 7.5 with 7 lM phosphopeptide Ias substrate. Partial amino-acid sequence determination ofthe purified porcine enzyme by MS revealed similaritywith a human sequence representing a human chromosome9 gene of hitherto unknown function. Molecularcloning from a human embryonic kidney cell cDNAlibraryfollowed by expression and purification, yielded aprotein with a molecular mass of 13 700 Da, and anEDTA-insensitive phosphohistidine phosphatase activityof 9 lmolÆmin)1Æmg)1 towards phosphopeptide I. Nodetectable activity was obtained towards a set of phosphoserine-,phosphothreonine-, and phosphotyrosine peptides.Northern blot analysis indicated that the humanphosphohistidine phosphatase mRNA was present preferentiallyin heart and skeletal muscle. These resultsprovide a new tool for studying eukaryotic histidinephosphorylation/dephosphorylation.
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| 3. |
- Ek, Pia, et al.
(författare)
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Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates phospholysine of chemically phosphorylated histone H1 and polylysine
- 2015
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Ingår i: Upsala Journal of Medical Sciences. - : Uppsala Medical Society. - 0300-9734 .- 2000-1967. ; 120:1, s. 20-27
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Tidskriftsartikel (refereegranskat)abstract
- Background. Phosphohistidine phosphatase 1 (PHPT1), also named protein histidine phosphatase (PHP), is a eukaryotic enzyme dephosphorylating proteins and peptides that are phosphorylated on a histidine residue. A preliminary finding that histone H1, which lacks histidine, was phosphorylated by phosphoramidate and dephosphorylated by PHPT1 prompted the present investigation. Methods. Histone H1 and polylysine were phosphorylated at a low concentration (3.9 mM) of phosphoramidate. Their dephosphorylation by recombinant human PHPT1 was investigated by using a DEAE-Sepharose spin column technique earlier developed by us for studies on basic phosphoproteins and phosphopeptides. Determination of protein-bound, acid-labile phosphate was performed by a malachite green method. Mass spectrometry (MS) was used to investigate the occurrence of N-epsilon-phospholysine residues in a phosphorylated histone H 1.2 preparation, and to measure the activity of PHPT1 against free N-omega-phosphoarginine. Results. Histone H1.2, which lacks histidine, was phosphorylated by phosphoramidate on several lysine residues, as shown by MS. PHPT1 was shown to dephosphorylate phosphohistone H1 at a rate similar to that previously described for the dephosphorylation of phosphohistidine-containing peptides. In addition, phosphopolylysine was an equally good substrate for PHPT1. However, no dephosphorylation of free phosphoarginine by PHPT1 could be detected. Conclusion. The finding that PHPT1 can dephosphorylate phospholysine in chemically phosphorylated histone H1 and polylysine demonstrates a broader specificity for this enzyme than known so far.
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| 4. |
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| 5. |
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| 6. |
- Adolfsson, Päivi, 1956-, et al.
(författare)
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Dietitians’ challenges when consulting to adults with intellectual disabilities
- 2019
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Ingår i: Tizard Learning Disability Review. - 1359-5474 .- 2042-8782. ; 24:4, s. 153-162
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: The purpose of this paper is to investigate registered dietitians' (RDs) experiences in consulting to adults with intellectual disabilities (ID) in Sweden.Design/methodology/approach: A descriptive study using a study-specific web-based questionnaire was administered, comprising both multiple-choice questions with a space for comments and open-ended questions. The open-ended answers and comments from 53 respondents were analysed with systematic text condensation.Findings: Four categories were identified: RDs' experiences from the first meeting; explanations for late initial contact; the actions taken by RDs; and necessary measures for more sustainable nutrition care. Ten sub-categories described the challenges that RDs experience in more detail.Practical implications: It is necessary to provide adults with ID and their supporting staff with individually tailored nutritional information. Individuals with ID must be actively involved in lifestyle changes that affect their everyday life. The RD must be included in the interdisciplinary team supporting adults with ID. If a new practice is to be implemented, it should be compatible with the existing values of adults with ID and their staff and must be feasible to implement in the everyday life of the individual.Originality/value: This paper identified several barriers that should be overcome in relation to the preparation of RDs for consultation with adults with ID about nutritional health issues. A systematic structure, knowledge about nutrition and knowledge about adults with ID and their living situations are needed. An assessment instrument may meet health promotion needs and facilitate longitudinal follow-ups of nutritional problems.
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| 7. |
- Adolfsson, Päivi, et al.
(författare)
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Dietitians’ endeavor to contribute to the nutritional health of children and youth with intellectual disability and autism
- 2023
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Ingår i: International Journal of Developmental Disabilities. - : Taylor & Francis. - 2047-3869.
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Tidskriftsartikel (refereegranskat)abstract
- The aim of the present study was to explore the experiences of registered dietitians (RD) who consult children and youth with intellectual disability (ID) and autism. Another aim was to investigate how knowledge and working methods were transferred to RDs working with adults with ID and autism. Twenty-six RDs completed a web-based study-specific questionnaire with multiple-choice and open-ended questions. The respondents’ comments and responses to the open-ended questions were analyzed using systematic text condensation. The analyses resulted in four categories: Reachability and accessibility of RDs, Clients do not comply with RDs’ dietary advice, RD finds individual solutions and Better collaboration for better knowledge. It was noteworthy that RDs’ undergraduate education did not prepare them for clients with ID and autism. Instead, they learned by doing and from other professionals at the clinic if they collaborate with them or were part in teams around the client. The RDs reported a lack of national routines for the transition process of nutrition support from young to adult.
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| 8. |
- Adolfsson, Päivi, 1956-, et al.
(författare)
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Significant others’ perspectives on experiences of meal-oriented support and diet counselling for adults with intellectual disabilities who live in supported housing
- 2024
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Ingår i: International Journal of Developmental Disabilities. - : Taylor & Francis. - 2047-3869 .- 2047-3877. ; 70:3, s. 435-443
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Tidskriftsartikel (refereegranskat)abstract
- The quality of meal-oriented support for people with intellectual disabilities is important for their health. The aim of the present study was to explore the experiences of meal-oriented support and diet counselling for adults with intellectual disabilities living in supported housing, from the perspective of housing staff and mothers. Five focus group interviews, including nine supporting staff members and nine mothers, were conducted. The interviews were analyzed using systematic text condensation. Five themes appeared; Extensive needs of the individual, Staff skills determine the food intake, Informal caregivers make up for shortage of support, Effective collaboration with a registered dietitian is needed and Responsibility of the organization state that professionalization of staff is needed. Lacking resources, such as time and nutritional knowledge, insufficient considerations of individual needs, and high staff turnover influence the meal-orientated services negatively. This study brings to the fore, staff working practices and the complexity of providing meal-oriented support for people with intellectual disabilities. Staff need skills to perform individually tailored support. This is best accomplished through effective collaboration between housing staff and relatives underpinned by knowledge from a registered dietitian. The working practices must be structured at the organizational level of the services.
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| 9. |
- Alexandridis, Vasileios, et al.
(författare)
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Retropubic slings are more efficient than transobturator at 10-year follow-up : a Swedish register-based study
- 2023
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Ingår i: International Urogynecology Journal. - : Springer Science and Business Media LLC. - 0937-3462 .- 1433-3023. ; 34:6, s. 1307-1315
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Tidskriftsartikel (refereegranskat)abstract
- Introduction and hypothesis: Long-term performance of mid-urethral slings (MUS) and potential differences between the retropubic and the transobturator technique for insertion are scarcely studied. This study aims to evaluate the efficacy and safety 10 years after surgery and compare the two main surgical techniques used. Methods: Women who underwent surgery with a MUS between 2006 and 2010 were identified using the Swedish National Quality Register of Gynecological Surgery and were invited 10 years after the operation to answer questionnaires regarding urinary incontinence and its impact on quality-of-life parameters (UDI-6, IIQ-7) and impression of improvement, as well as questions regarding possible sling-related complications and reoperation. Results: The subjective cure rate reported by 2421 participating women was 63.3%. Improvement was reported by 79.2% of the participants. Women in the retropubic group reported higher cure rates, lower urgency urinary incontinence rates and lower UDI-6 scores. No difference was shown between the two methods regarding complications, reoperation due to complications or IIQ-7 scores. Persisting sling-related symptoms were reported by 17.7% of the participants, most commonly urinary retention. Mesh exposure was reported by 2.0%, reoperation because of the tape by 5.6% and repeated operation for incontinence by 6.9%, significantly more in the transobturator group (9.1% vs. 5.6%). Preoperative urinary retention was a strong predictor for impaired efficacy and safety at 10 years. Conclusions: Mid-urethral slings demonstrate good results for the treatment of stress urinary incontinence and tolerable complication profiles in a 10-year perspective. The retropubic approach displays higher efficacy than the transobturator, with no difference regarding safety.
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| 10. |
- Beckman Sundh, Ulla, 1953-
(författare)
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Studies on Phosphohistidine Phosphatase 1 : What? Where? Why?
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Phosphohistidine phosphatase 1 (PHPT1) is a small protein, consisting of 125 amino acids, that catalyzes the dephosphorylation of histidine but does not have any activity towards other phosphorylated amino acids. PHPT1 was identified in 2002, and is so far the only mammalian histidine phosphatase known, but still little is known about its physiological role. No mammalian histidine kinases have hitherto been identified.Phosphorylation is one of the most important ways in which the structure and activity of a protein may be changed after translation. Proteins are phosphorylated on the side chain of amino acid residues. When a hydroxyl is phosphorylated the result is a phosphoester and when a nitrogen is phosphorylated the result is a phosphoamidate. Histidine may be phosphorylated on either of the two nitrogens of the imidazole ring of the side chain. The resulting phosphoamidate bond is labile and rich in energy, which makes histidine phosphorylation highly reversible and flexible. However, histidine phosphorylation is less studied than that of the phosphoesters due to the acid lability of the phosphoamidate bond.The work described in this thesis was focused on further elucidating the physiological role of PHPT1. Amino acid residues of importance for the activity of PHPT1 were identified, and mutants with decreased phosphatase activity were produced. These mutants have been used in studies on the function of PHPT1. By using immunohistochemical methodology the localization of PHPT1 in both mouse and human tissues was determined, with mainly similar results. A general finding was that expression of PHPT1 was high in epithelial cells with short turnover time, indicating that PHPT1 may have an important role in proliferating cells. We have also developed a comparatively fast and simple screening method for determination of PHPT1 activity. Since research in this field has been hampered by the lack of efficient and practical methodology, hopefully this new method will be an asset in search of inhibitors for PHPT1, which in turn may be used for detection of the elusive mammalian histidine kinases, the finding of which may give major breakthroughs in the field.
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| 11. |
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| 12. |
- Dahlqvist-Edberg, Ulla, et al.
(författare)
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Purification of a Ca2+-activated protease from rat erythrocytes and its possible effect on pyruvate kinase in vivo
- 1981
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Ingår i: Biochimica et Biophysica Acta-Enzymology. - : Elsevier BV. - 0005-2744. ; 660:1, s. 96-101
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Tidskriftsartikel (refereegranskat)abstract
- A Ca2+-activated protease with [32P]phosphopyruvate kinase as substrate was purified to about 50% from rat erythrocytes. The purification involved chromatography on Sepharose/Sephadex gels, DEAE-cellulose and (NH4)2SO4 precipitation. The protease required 3.3 mM Ca2+ for full activity. When pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) was purified from erythrocytes incubated with [32P]phosphate it contained 0.5 mol [32P]phosphate/mol enzyme subunit. When 3.3 mM Ca2+ were added at hemolysis this incorporation decreased. The possible importance of this Ca2+-activated protease for the regulation of pyruvate kinase in erythrocytes is discussed.
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| 13. |
- Dahlqvist-Edberg, Ulla, et al.
(författare)
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The demonstration in rat liver cell sap of protein kinase and phosphoprotein phosphatase active on fructose-bisphosphatase
- 1982
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Ingår i: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology. - : Elsevier BV. - 0167-4838 .- 1879-2588. ; 706:2, s. 239-244
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Tidskriftsartikel (refereegranskat)abstract
- A protein kinase active on fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) was demonstrated in rat liver cell sap. The protein kinase activity was stimulated by cyclic AMP and coincided with the activity of cyclic AMP-dependent protein kinase type I. In addition, three different peaks of phosphoprotein phosphatase active on [32P] phosphofructose-bisphosphatase were found on chromatography of rat liver cell sap on a DEAE-cellulose column. These phosphatases needed divalent cations for full activity. 5'-AMP, a negative modulator of fructose-bisphosphatase, had no effect on the phosphorylation-de-phosphorylation reactions of the enzyme. ATP and Ca2+ did not influence the dephosphorylation reaction of fructose-bisphosphatase.
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| 14. |
- Dahlqvist, Ulla, et al.
(författare)
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Endogenous substrates of protein kinase in rat liver cell sap under different dietary conditions
- 1978
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0304-4165. ; 540:1, s. 13-23
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Tidskriftsartikel (refereegranskat)abstract
- Liver cell sap from normally fed rats, rats fed with a high-carbohydrate diet and fasted rats was chromatographed on DEAE-cellulose (pH 7.0). The chromatogram from each diet group was analyzed for pyruvate kinase activity and endogenous substrates of cyclic AMP-stimulated protein kinase. The materials were pooled into five phosphorylatable fractions, in each of which phosphate incorporation at 0.1 mM and 1.0 mM [32P]ATP in the presence of cyclic AMP and protein kinase was determined. For characterization of the phosphorylatable components, thin-layer gel chromatography on Sephadex G-200 and polyacrylamide gel electrophoresis in detergent were used for determination of native and minimal molecular weights, respectively. Except for pyruvate kinase, eight components which incorporated at least 0.05 nmol of [32P]phosphate/g of liver were detected. The phosphorylation of four of them was stimulated by cyclic AMP. Their minimal molecular weights were 42000, 21000, 52000 and 49000. The component with a minimal molecular weight of 42000 seemed to have a native molecular weight of 160000. Both the 21000 and the 52000 component had a native molecular weight of about 110000-120000. The protein with a minimal molecular weight of 49000 could not be correlated with certainty to a native molecular weight. The proteins whose phosphorylation was not stimulated by cyclic AMP had minimal molecular weights of 54000, 39000, 34000 and 22000.
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| 15. |
- Edlund, Bror, et al.
(författare)
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Amino acid sequence at the phosphorylated site of rat liver pyruvate kinase
- 1975
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - 0006-291X .- 1090-2104. ; 67:4, s. 1516-1521
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Tidskriftsartikel (refereegranskat)abstract
- One dominating peptic phosphopeptide, Asx-Thr-Lys-Gly-Pro-Glx-Ile-Glx-Thr-Gly-Val-Leu-Arg-Arg-Ala-(32P)SerP-Val-Ala-Glx-Leu, was obtained from rat liver pyruvate kinase (type L) phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase from the same tissue. The sequence around the phosphorylated serine residue is similar to that of a corresponding but smaller peptic phosphopeptide previously isolated from pig liver (type L) pyruvate kinase, Leu-Arg-Arg-Ala-(32P)SerP-Leu.
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| 16. |
- Ek, Pia, et al.
(författare)
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Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3´, 5´-AMP-stimulated protein kinase
- 1976
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 429:2, s. 374-382
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Tidskriftsartikel (refereegranskat)abstract
- The kinetics of rat liver L-type pyruvate kinase (EC 2.7.1.40), phosphorylated with cyclic AMP-stimulated protein kinase from the same source, and the unphosphorylated enzyme have been compared. The effects of pH and various concentrations of substrates, Mg2+, K+ and modifiers were studied. In the absence of fructose 1, 6-diphosphate at pH 7.3, the phosphorylated pyruvate kinase appeared to have a lower affinity for phosphoenolpyruvate (K0.5=0.8 mM) than the unphosphorylated enzyme (K0.5=0.3 mM). The enzyme activity vs. phosphoenolpyruvate concentration curve was more sigmoidal for the phosphorylated enzyme with a Hill coefficient of 2.6 compared to 1.6 for the unphosphorylated enzyme. Fructose 1, 6-diphosphate increased the apparent affinity of both enzyme forms for phosphoenolpyruvate. At saturating concentrations of this activator, the kinetics of both enzyme forms were transformed to approximately the same hyperbolic curve, with a Hill coefficient of 1.0 and K0.5 of about 0.04 mM for phosphoenolpyruvate. The apparent affinity of the enzyme for fructose 1, 6-diphosphate was high at 0.2 mM phosphoenolpyruvate with a K0.5=0.06 muM for the unphosphorylated pyruvate kinase and 0.13 muM for the phosphorylated enzyme. However, in the presence of 0.5 mM alanine plus 1.5 mM ATP, a higher fructose 1, 6-diphosphate concentration was needed for activation, with K0.5 of 0.4 muM for the unphosphorylated enzyme and of 1.4 muM for the phosphorylated enzyme. The results obtained strongly indicate that phosphorylation of pyruvate kinase may also inhibit the enzyme in vivo. Such an inhibition should be important during gluconeogenesis.
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| 17. |
- Ek, Pia, et al.
(författare)
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Exogenous protein kinases A and C, but not endogenous prostasome-associated protein kinase, phosphorylate semenogelins I and II from human semen
- 2002
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Ingår i: Journal of Andrology. - 0196-3635 .- 1939-4640. ; 23:6, s. 806-814
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Tidskriftsartikel (refereegranskat)abstract
- Semenogelins I and II are the quantitatively dominating proteinsin humansemen. They comprise the major part of the sperm-entrappinggel formed atejaculation, which subsequently liquefies dueto proteolysis of thegel-forming proteins by prostate-specificantigen (PSA). The mechanism behindgel formation and its physiologicalsignificance is not known. We have studiedphosphorylation anddephosphorylation of human semenogelins. Both werephosphorylatedby protein kinases A and C (PKA and PKC, respectively) at arateabout 5 times less than that of histone. For PKA, incorporated(32P)phosphateinto semenogelin approached a maximum above 1mol/mol. Correspondingvalues for phosphorylation of the semenogelins with PKCweregreater than 10. There was no change in the sensitivity ofphosphosemenogelinsto proteolysis by PSA. Serine (PKA) and serine andthreonine(PKC) were the phosphate-accepting amino acid residues, andallincorporated (32P)phosphate could be removed from the semenogelinswithhuman acid phosphatase. Nil or very little phosphate could bedetected inpurified semenogelins isolated from seminal plasma.In vivo, about half theprotein kinase activity in seminal plasmawas bound to prostasomes. PKA butnot PKC purified from prostasomescould phosphorylate specific substrates, butthey could phosphorylateeither of the semenogelins.
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| 18. |
- Ek, Pia, et al.
(författare)
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The kinetics of unphosphorylated, phosphorylated and proteolytically modified fructose bisphosphatase from rat liver
- 1981
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Ingår i: Biochimica et Biophysica Acta. - 0005-2744. ; 662:2, s. 265-270
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Tidskriftsartikel (refereegranskat)abstract
- Phosphorylation of fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) by the catalytic subunit of cyclic AMP-dependent protein kinase from pig muscle decreased the K0.5 for fructose-bisphosphate from 21 to 11 microM. When the phosphorylated fructose-bisphosphatase was treated with trypsin the K0.5 increased to 22 microM. The K0.5 also increased when the phosphoenzyme was treated with a partially purified phosphatase from rat liver. There was no difference between the unphosphorylated and phosphorylated enzyme with respect to pH dependence, the pH optimum being about 7.0 for both. Limited treatment of fructose-bis-phosphatase with subtilisin, which cleaves the enzyme at its unphosphorylatable N-terminal part, increased the pH optimum more than limited treatment with trypsin, which releases the phosphorylated peptide at the C-terminal part of fructose-bisphosphatase. The phosphorylated site on the phosphorylated fructose-bisphosphatase was more easily split off by trypsin treatment than the corresponding unphosphorylated site. The results suggest in addition to the glucagon-induced phosphorylation of fructose-bisphosphatase described by Claus et al. [1] that the phosphorylation-dephosphorylation of fructose-bisphosphatase could be of importance for the hormonal regulation of the enzyme in vivo.
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| 19. |
- Ekman, Pia, et al.
(författare)
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The quantity of protein-bound (32P)phosphotyrosine in hepatocytes and fibroblasts : The effects of tyrosine protein kinase activating agents
- 1987
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Ingår i: Journal of Biochemistry (Tokyo). - 0021-924X .- 1756-2651. ; 101:4, s. 863-870
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Tidskriftsartikel (refereegranskat)abstract
- Tyrosine protein kinase activities have been demonstrated in transformed and normal cell systems. So far, few data on the quantity of protein-bound phosphotyrosine in intact cells have been published. A knowledge of the stoichiometric increase in phosphotyrosine in cells after hormonal induction could be of interest when evaluating the importance of the tyrosine protein kinase activities found. By the addition of a known amount of unlabeled phosphotyrosine to the precipitated protein of 32P-phosphate-labeled cells it was possible after alkaline hydrolysis to spectrophotometrically follow the phosphotyrosine during consecutive chromatographies of the material. From the specific radioactivity of the purified phosphotyrosine the initial concentration of [32P]phosphotyrosine could be calculated. The method proved to be useful for the determination of [32P]phosphotyrosine is small amounts of cells. The minimum detectable amount of [32P]phosphotyrosine was about 1 pmol, and as an example, only 2.5 X 10(6) fibroblasts were needed. By this method it was shown that platelet-derived growth factor increased protein-bound [32P]phosphotyrosine from 600 to 3,200 pmol/g of fibroblasts, while insulin only increased the [32P]phosphotyrosine from 110 to 120 pmol/g of hepatocytes.
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| 20. |
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| 21. |
- Hellström, Pia, 1960-
(författare)
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Fenton Pre-treatment of a Birch Kraft Pulp for MFC preparation
- 2015
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The potential to use acidic hydrogen peroxide in the presence of ferrous ions (Fenton’s reagent) as a pre-treatment when producing microfibrillar cellulose (MFC) from a fully bleached birch (Betula verucosa) kraft pulp was investigated and the properties of the produced MFC was compared to the properties of a MFC produced with enzymatic pre-treatment with a monocomponent endoglucanase (FiberCare® R). The mechanical treatment to MFC was performed in a laboratory colloid mill or in a pilot high-pressure homogeniser and the pre-treated pulps as well as the produced MFCs were chemically and morphologically characterised. Additionally, the MFCs produced in the colloid mill were evaluated as strength enhancers in test sheets representing the middle ply of paperboard.From the chemical characterisation, it was concluded that the Fenton pre-treatment caused a decrease in the degree of polymerisation (DP) and an increase in both carboxyl- and carbonyl groups. The increase in carbonyl groups could not be explained by the formation of new reducing end groups due to depolymerisation which indicates that carbonyl groups are introduced along the cellulose chain. The enzymatic pre-treatment as performed in this study caused less impact on the cellulosic material, i.e. resulted in a pulp with a higher DP and a much lower amount of carbonyl- and carboxylic groups compared with the Fenton pre-treated pulps. In the subsequent mechanical treatment in a colloid mill, the Fenton pre-treated pulps were easier to process mechanically i.e. reached a higher specific surface area and a higher surface charge at a given mechanical treatment time compared to enzymatic pre-treated pulps and pulps not subjected to any pre-treatment. These findings were confirmed when MFCs were produced by homogenisation at high pressure in multiple passes; the birch kraft pulp was either pre-treated with Fenton’s reagent or the combined mechanic and enzymatic pre-treatment methodology used at the Centre Technique du Papier (CTP, France). By size fractionation, rheological measurements and scanning electron microscopy, it was revealed that Fenton pre-treatment resulted in MFC suspension containing a significantly higher proportion of small sized material (< 0.2 mm).When the MFCs were evaluated as strength enhancers in test sheets produced from a furnish consisting of a spruce (Picea abies) chemithermomechanical pulp, MFC and a retention system containing cationic starch and an anionic silica sol, Fenton pre-treated MFCs increased the strength properties more than the enzymatic pre-treated MFCs. Addition of 5 wt% Fenton pre-treated MFC resulted in an increase in z-directional strength of about 50%, an increase in tensile stiffness index of about 25% and an increase in tensile index of 35% compared to test sheets prepared without MFC addition.
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| 22. |
- Humble, Elisabet, et al.
(författare)
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Amino acid sequence at the phosphorylated site of rat liver fructose-1,6-diphosphatase and phosphorylation of a corresponding synthetic peptide
- 1979
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - 0006-291X .- 1090-2104. ; 90:3, s. 1064-1072
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Tidskriftsartikel (refereegranskat)abstract
- Rat liver fructose-1,6-diphosphatase was phosphorylated with (32P)ATP and the catalytic subunit of cyclic AMP-dependent protein kinase from pig muscle. After digestion with pepsin, α-chymotrypsin and subtilisin a peptide with the amino-terminal sequence Ser-Arg-Tyr-(32P)SerP-Leu-Pro-Leu-Pro was isolated. A synthetic unphosphorylated heptapeptide with the same amino acid sequence, ending with leucine, was phosphorylated with an apparent Km of 400 μM, while the apparent Km value for fructose-1,6-diphosphatase was 30 μM (subunit concentration). The Vmax value was 20 times higher for the peptide than for the enzyme.
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| 23. |
- Inturi, Raviteja, 1985-, et al.
(författare)
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A splice variant of the human phosphohistidine phosphatase 1 (PHPT1) is degraded by the proteasome
- 2014
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Ingår i: International Journal of Biochemistry and Cell Biology. - : Elsevier. - 1357-2725 .- 1878-5875. ; 57, s. 69-75
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Tidskriftsartikel (refereegranskat)abstract
- Regulation of protein activity by phosphorylation is central in many cellular processes. Phosphorylation of serine, threonine and tyrosine residues is well documented and studied. In addition, other amino acids, like histidine can be phosphorylated, but neither the mechanism nor the function of this modification is well understood. Nevertheless, there is a 14 kDa enzyme with phosphohistidine phosphatase activity, named PHPT1, found in most animals, but not in bacteria, plant or fungi. There are a few splice variant transcripts formed from the human PHPT1 locus and some of them are predicted to form variant proteins, but studies of these proteins are lacking. In order to get insight into the possible function of the variant transcripts encoded at the PHPT1 locus, ectopic expression of PHPT1 transcript variant 6, predicted to be degraded by the non-sense mediated mRNA decay pathway, in HeLa cells was undertaken. In HeLa cells the splice variant protein was degraded by the proteasome, unlike the wild type protein. Using an in silico modeling approach the variant C-terminal end of the proteins were predicted to form different secondary structures that might explain the different properties of the two proteins. The specific degradation of the PHPT1 splice variant indicates that at least for the PHPT1 protein, the quality control and the self-guarding of the cellular system works at two levels, first at the RNA level, aberrant transcripts are degraded by the non-sense mediated mRNA decay pathway, and the small amount of proteins that are formed will be degraded by the proteasome.
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| 24. |
- Järv, Jaak, et al.
(författare)
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Quantitative structure-activity relationships in protein kinase C reaction with synthetic peptides derived from myelin basic protein
- 1996
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Ingår i: Bioorganic chemistry (Print). - : Elsevier BV. - 0045-2068. ; 24:2, s. 159-168
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Tidskriftsartikel (refereegranskat)abstract
- A set of peptides, Lys-Arg-Pro-Ser-X-Arg-Ala-Lys-Ala, where X stands for Ala, Val, Leu, Ile, Phe, Pro, Lys, Arg, Asp, Glu, Asn, Gln, and His, was synthesized and the kinetics of their phosphorylation by protein kinase C was studied. All compounds, except the peptide with Pro at the position X, were effectively phosphorylated by this enzyme, and for these substrates the kinetic constantsKm, maximal velocity constantsV, and second-order rate constantskIIwere determined. The data were analyzed by means of quantitative structure–activity relationships, taking into account hydrophobicity of the variable amino acids, bulkiness of their side groups quantified by molecular refractivity constants MR, and ionic status of these substituents by using an independent variable +1 for cationic, −1 for anionic, and 0 for nonionic substituents. These structural factors influenced theKmvalues, while the maximal velocity of phosphorylation depended mostly on the ionic status of the variable amino acid. The latter effect seems to characterize electrostatic interaction between the substrate molecule and some negative charge located in the enzyme active center.
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| 25. |
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| 26. |
- Lundmark Drca, Anna, et al.
(författare)
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Dyspareunia and pelvic pain: comparison of mid-urethral sling methods 10 years after insertion
- 2024
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Ingår i: International Urogynecology Journal. - 1433-3023.
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Tidskriftsartikel (refereegranskat)abstract
- Introduction and hypothesisThe mid-urethral sling (MUS) has been used for more than 30 years to cure stress urinary incontinence. The objective of this study was to assess whether surgical technique affects the outcome after more than ten years, regarding dyspareunia and pelvic pain.MethodsIn this longitudinal cohort study we used the Swedish National Quality Register of Gynecological Surgery to identify women who underwent MUS surgery in the period 2006–2010. Out of 4348 eligible women, 2555 (59%) responded to the questionnaire sent out in 2020–2021. The two main surgical techniques, the retropubic and the obturatoric approach, were represented by 1562 and 859 women respectively. The Urogenital Distress Inventory-6 (UDI-6) and the Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire (PISQ-12), as well as general questions concerning the MUS surgery, were sent out to the study population. Dyspareunia and pelvic pain were defined as primary outcomes. Secondary outcomes included PISQ-12, general satisfaction, and self-reported problems due to sling insertion.ResultsA total of 2421 women were included in the analysis. Among these, 71% responded to questions regarding dyspareunia and 77% responded to questions regarding pelvic pain. In a multivariate logistic regression analysis of the primary outcomes, we found no difference in reported dyspareunia (15% vs 17%, odds ratio (OR) 1.1, 95% CI 0.8–1.5) or in reported pelvic pain (17% vs 18%, OR 1.0, 95% CI 0.8–1.3) between the retropubic and obturatoric techniques among study responders.ConclusionDyspareunia and pelvic pain 10–14 years after insertion of a MUS do not differ with respect to surgical technique.
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| 27. |
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| 28. |
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| 29. |
- Marknell DeWitt, Åsa, 1966-
(författare)
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Use of Recombinant Allergens for Component-Resolved Diagnostics (CRD) in IgE-Mediated Allergy
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Immunoglobulin E (IgE)-mediated allergy occurs when our immune system causes a reaction to otherwise harmless substances (allergens). Allergens are predominantly proteins present in biological materials such as pollens, mites, animal epithelia, moulds and foods. In vitro tests for specific IgE antibodies usually employ an allergen source extract as an antibody capturing reagent. The proportion of allergenic molecules in these biochemically complex extracts may vary.Recombinant allergens may be obtained in large quantities with biotechnological techniques. These proteins can be characterized biochemically and immunologically, resulting in tests with minimal batch-to-batch variation. This thesis describes different uses of recombinant allergens in component-resolved diagnostics (CRD).In CRD, single allergenic proteins are used to establish a sensitization profile of the patient. Two timothy grass (Phleum pratense) pollen allergens, Phl p 11 and Phl p 4, were cloned and expressed as recombinant proteins. They were subsequently characterized and can, for example, be used in a panel for grass pollen CRD.Single allergens may be useful as diagnostic markers for allergic sensitization. This phenomenon was studied using tropomyosin, a major allergen from the shrimp Penaeus aztecus (Pen a 1). The characteristics of the recombinant and natural proteins were compared. The recombinant tropomyosin was then extensively tested using specific competition for IgE binding against extracts of other crustacean species, house dust mite and cockroach.In cases when an important allergen is missing or underrepresented in a natural extract, the corresponding recombinant allergen may be added to the extract as a spiking reagent. Previous studies have shown that latex extracts for diagnostic testing may lack the allergen Hev b 5. Recombinant Hev b 5 was expressed from a synthetic gene construct, incorporating several adaptations to enable efficient large scale production of the recombinant protein, to be used as a spiking reagent.
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| 30. |
- Prasthofer, T, et al.
(författare)
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Protein kinase C phosphorylates two of the four known syndecan cytoplasmic domains in vitro
- 1995
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Ingår i: Biochemistry and Molecular Biology International. - 1039-9712. ; 36:4, s. 793-802
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Tidskriftsartikel (refereegranskat)abstract
- The transmembrane heparan sulfate proteoglycans of the syndecan family are implicated to participate in several cellular reactions which are dependent on protein kinase C. We have used an in vitro assay to assess whether any of the Peptides corresponding to the complete cytoplasmic domains of rat syndecans 1 through 4 were used as substrates for the enzyme. The syndecan-2 (fibroglycan) and syndecan-3 (N-syndecan) peptides were both found to be phosphorylated by protein kinase C with Kms of 15 +/- 3 microM and 85 +/- 25 microM, respectively, while the syndecan-1 and -4 peptides were not phosphorylated under the conditions used. The sites of in vitro phosphorylation for syndecans-2 and -3 were localized to ser-197 and ser-339, respectively. Thus, among 13 available sites (serines and threonines) in the four peptides, two were selectively modified by the enzyme. The specificity and the kinetics of the reactions indicate that the cytoplasmic domains of syndecan-2 and -3 are likely to be physiological substrates for protein kinase C.
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| 31. |
- Springett, Jane, 1952-, et al.
(författare)
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Annual report 2004
- 2005
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Rapport (övrigt vetenskapligt/konstnärligt)
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| 32. |
- Springett, Jane, et al.
(författare)
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Annual report 2004
- 2005
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Bok (övrigt vetenskapligt/konstnärligt)
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| 33. |
- Tavoosidana, Gholamreza, et al.
(författare)
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Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer
- 2011
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:21, s. 8809-8814
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Tidskriftsartikel (refereegranskat)abstract
- Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal fluid, to appear in interstitial space and therewith into peripheral circulation. The suitability of prostasomes as blood biomarkers in patients with prostate cancer was tested by using an expanded variant of the proximity ligation assay (PLA). We developed an extremely sensitive and specific assay (4PLA) for detection of complex target structures such as microvesicles in which the target is first captured via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA strands. The requirement for coincident binding by five antibodies to generate an amplifiable reporter results in both increased specificity and sensitivity. The assay successfully detected significantly elevated levels of prostasomes in blood samples from patients with prostate cancer before radical prostatectomy, compared with controls and men with benign biopsy results. The medians for prostasome levels in blood plasma of patients with prostate cancer were 2.5 to sevenfold higher compared with control samples in two independent studies, and the assay also distinguished patients with high and medium prostatectomy Gleason scores (8/9 and 7, respectively) from those with low score (<= 6), thus reflecting disease aggressiveness. This approach that enables detection of prostasomes in peripheral blood may be useful for early diagnosis and assessment of prognosis in organ-confined prostate cancer.
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| 34. |
- Tesi, Bianca, et al.
(författare)
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Diagnostic yield and clinical impact of germline sequencing in children with CNS and extracranial solid tumors : a nationwide, prospective Swedish study
- 2024
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Ingår i: The Lancet Regional Health. - : Elsevier. - 2666-7762. ; 39
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundChildhood cancer predisposition (ChiCaP) syndromes are increasingly recognized as contributing factors to childhood cancer development. Yet, due to variable availability of germline testing, many children with ChiCaP might go undetected today. We report results from the nationwide and prospective ChiCaP study that investigated diagnostic yield and clinical impact of integrating germline whole-genome sequencing (gWGS) with tumor sequencing and systematic phenotyping in children with solid tumors.MethodsgWGS was performed in 309 children at diagnosis of CNS (n = 123, 40%) or extracranial (n = 186, 60%) solid tumors and analyzed for disease-causing variants in 189 known cancer predisposing genes. Tumor sequencing data were available for 74% (227/309) of patients. In addition, a standardized clinical assessment for underlying predisposition was performed in 95% (293/309) of patients.FindingsThe prevalence of ChiCaP diagnoses was 11% (35/309), of which 69% (24/35) were unknown at inclusion (diagnostic yield 8%, 24/298). A second-hit and/or relevant mutational signature was observed in 19/21 (90%) tumors with informative data. ChiCaP diagnoses were more prevalent among patients with retinoblastomas (50%, 6/12) and high-grade astrocytomas (37%, 6/16), and in those with non-cancer related features (23%, 20/88), and ≥2 positive ChiCaP criteria (28%, 22/79). ChiCaP diagnoses were autosomal dominant in 80% (28/35) of patients, yet confirmed de novo in 64% (18/28). The 35 ChiCaP findings resulted in tailored surveillance (86%, 30/35) and treatment recommendations (31%, 11/35).InterpretationOverall, our results demonstrate that systematic phenotyping, combined with genomics-based diagnostics of ChiCaP in children with solid tumors is feasible in large-scale clinical practice and critically guides personalized care in a sizable proportion of patients.
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| 35. |
- Tesi, Bianca, et al.
(författare)
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Diagnostic yield and clinical impact of germline sequencing in children with CNS and extracranial solid tumors : a nationwide, prospective Swedish study
- 2024
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Ingår i: The Lancet Regional Health. - : Elsevier. - 2666-7762. ; 39
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Tidskriftsartikel (refereegranskat)abstract
- Background: Childhood cancer predisposition (ChiCaP) syndromes are increasingly recognized as contributing factors to childhood cancer development. Yet, due to variable availability of germline testing, many children with ChiCaP might go undetected today. We report results from the nationwide and prospective ChiCaP study that investigated diagnostic yield and clinical impact of integrating germline whole-genome sequencing (gWGS) with tumor sequencing and systematic phenotyping in children with solid tumors.Methods: gWGS was performed in 309 children at diagnosis of CNS (n = 123, 40%) or extracranial (n = 186, 60%) solid tumors and analyzed for disease-causing variants in 189 known cancer predisposing genes. Tumor sequencing data were available for 74% (227/309) of patients. In addition, a standardized clinical assessment for underlying predisposition was performed in 95% (293/309) of patients.Findings: The prevalence of ChiCaP diagnoses was 11% (35/309), of which 69% (24/35) were unknown at inclusion (diagnostic yield 8%, 24/298). A second-hit and/or relevant mutational signature was observed in 19/21 (90%) tumors with informative data. ChiCaP diagnoses were more prevalent among patients with retinoblastomas (50%, 6/12) and high-grade astrocytomas (37%, 6/16), and in those with non-cancer related features (23%, 20/88), and ≥2 positive ChiCaP criteria (28%, 22/79). ChiCaP diagnoses were autosomal dominant in 80% (28/35) of patients, yet confirmed de novo in 64% (18/28). The 35 ChiCaP findings resulted in tailored surveillance (86%, 30/35) and treatment recommendations (31%, 11/35).Interpretation: Overall, our results demonstrate that systematic phenotyping, combined with genomics-based diagnostics of ChiCaP in children with solid tumors is feasible in large-scale clinical practice and critically guides personalized care in a sizable proportion of patients.Funding: The study was supported by the Swedish Childhood Cancer Fund and the Ministry of Health and Social Affairs.
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| 36. |
- Trojanek, Joanna, et al.
(författare)
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Phosphorylation of plant proteins and the identification of protein - tyrosine kinase activity in maize seedlings
- 1996
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 235:1-2, s. 338-344
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Tidskriftsartikel (refereegranskat)abstract
- Phosphotyrosine was found to be 0.5% of the total phosphoamino acids labelled with [32P]orthophosphate in endogenous maize seedlings proteins. Two peaks of protein kinase activity towards phosphorylation of synthetic peptide poly (Glu80, Tyr20) were obtained after chromatography of protein extract of dark-grown etiolated maize seedlings on phosphocellulose. The phosphorylation of synthetic peptide as well as endogenous proteins was strongly stimulated by Mn2+. At least three endogenous proteins with molecular masses in the range of 40-65 kDa were predominantly phosphorylated. This phosphorylation was resistant to alkali treatment. Chemical, immunological and enzymatic data indicated the presence of tyrosine kinase activity and also phosphotyrosine in proteins of maize seedlings. The plant enzyme(s) is reminiscent known mammalian cytosolic tyrosine kinase(s).
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| 37. |
- Zhang, X-Q, et al.
(författare)
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Immunohistochemical localization of phosphohistidine phosphatase PHPT1 in mouse and human tissues
- 2009
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Ingår i: Upsala Journal of Medical Sciences. - : Uppsala Medical Society. - 0300-9734 .- 2000-1967. ; 114:2, s. 65-72
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Tidskriftsartikel (refereegranskat)abstract
- Protein histidine phosphorylation accounts for about 6% of the total protein phosphorylation in eukaryotic cells; still details concerning histidine phosphorylation and dephosphorylation are limited. A mammalian 14-kDa phosphohistidine phosphatase, also denominated PHPT1, was found 6 years ago that provided a new tool in the study of phosphohistidine phosphorylation. The localization of PHPT1 mRNA by Northern blot analysis revealed high expression in heart and skeletal muscle. The main object of the present study was to determine the PHPT1 expression on protein level in mouse tissues in order to get further information on the physiological role of the enzyme. Tissue samples from adult mice and 14.5-day-old mouse embryos were processed for immunostaining using a PHPT1-specific polyclonal antibody. The same antibody was also provided to the Swedish human protein atlas project (HPR) (http://www.proteinatlas.org/index.php). The results from both studies were essentially consistent with the previously reported expression of mRNA of a few human tissues. In addition, several other tissues, including testis, displayed a high protein expression. A salient result of the present investigation was the ubiquitous expression of the PHPT1 protein and its high expression in continuously dividing epithelial cells.
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