| 1. |
- Berts, Alf, et al.
(författare)
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Suppression of Ca2+ oscillations in glucagon-producing alfa2-cells by insulin/glucose and amino acids
- 1996
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 1310:2, s. 212-216
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Tidskriftsartikel (refereegranskat)abstract
- The cytoplasmic Ca2+ concentration ([Ca2+]i) was continuously monitored in single glucagon-producing α2-cells isolated from the mouse pancreas and later identified by immunostaining. Up to 60% of the α2-cells exhibited spontaneous [Ca2+]i oscillations (frequency 0.1–0.3/min) in a medium containing 3 mM glucose. In originating from a basal level of 60–100 nM, reaching peak values of 300–400 nM and promptly disappearing after blocking voltage-dependent Ca2+ channels with methoxyverapamil, the oscillations resembled those in insulin-releasing β-cells stimulated by glucose. The oscillatory activity was suppressed when combining elevation of glucose to 20 mM with the addition of 2–2000 ng/ml insulin. Whereas 10 mM of l-arginine or l-glycine transformed the oscillations into sustained elevation of [Ca2+];, there was no response to 1 mM tolbutamide or 0.1–1 mM γ-aminobutyric acid. The observations that α2-cells differ from islet cells secreting insulin and somatostatin in responding to adrenaline with mobilisation of intracellular calcium can be used for their rapid identification. It is suggested that the oscillations reflect periodic entry of Ca2+ due to variations of the membrane potential.
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| 2. |
- Bhalerao, RP, et al.
(författare)
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Structure and energy-transfer of the phycobilisome in a linker protein replacement mutant of cyanobacterium synechococcus-7942
- 1991
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 1060:1, s. 59-66
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Tidskriftsartikel (refereegranskat)abstract
- The role of the linker proteins in the biogenesis and energy transfer of the phycobilisome rod was monitored by making insertional inactivation in the cpcI gene coding for the core-proximal 33 kilodalton (kDa) protein in the cyanobacterium Synechococcus 7942. The insertion leaves the cpcH gene coding for the core-distal 30 kDa protein intact and functional. Analysis of the phycobilisome protein composition of the cpcI mutant shows that the 30 kDa protein is present in normal amounts in the rod, indicating that the 30 kDa linker protein can replace the 33 kDa protein in the biogenesis and structural integrity of the rod. The absorption and fluorescence characteristics of the mutated phycobilisome is almost indistinguishable from that of the wild-type of the same rod length. The fluorescence kinetics from the cpcI mutant show that the dominating decay component has a lifetime from phycocyanin of 69 ps as compared to 72 ps found for the wild-type phycobilisome with the same rod length. The results show that replacing the 33 kDa for the 30 kDa linker in the rod does not alter the energy harvesting or the energy transfer characteristics of the rod in contrast to what has been concluded from data obtained from in vitro experiments. We conclude that the linker polypeptides have only a minor influence on the energy transfer characteristics of the rod but are mainly involved in determining the length of the rod in response to changing environmental light conditions.
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| 3. |
- Birnir, Bryndis, et al.
(författare)
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Expression and characterization of the intestinal Na+/glucose cotransporter in COS-7 cells.
- 1990
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 1048:1, s. 100-4
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Tidskriftsartikel (refereegranskat)abstract
- Cells derived from the simian kidney, COS-7 cells, were transfected with a eucaryotic expression vector (pEUK-C1) containing the clone for the rabbit intestinal Na+/glucose cotransporter. Expression was monitored after transfection with lipofectin by measuring the initial rate of alpha-methylglucopyranoside (MeGlc) uptake. Cells transfected with vector containing the cDNA for the Na+/glucose cotransporter expressed Na(+)-dependent MeGlc transport. Neither control cells nor cells transfected with vector lacking cloned cDNA expressed the cotransporter. Na(+)-dependent MeGlc uptake into transfected cells was saturable (Km 150 microM), phlorizin-sensitive (Ki 11 microM), and inhibited by sugar analogs (D-glucose greater than MeGlc greater than D-galactose greater than 3-O-methyl-D-glucoside greater than D-allose much greater than L-glucose). Europium was able to mimic Na+ in driving MeGIC uptake. Finally, tunicamycin, an inhibitor of asparagine-linked glycosylation, inhibited the expression of Na(+)-dependent MeGlc transport 80%. We conclude that the rabbit intestinal Na+/glucose cotransporter expressed in COS-7 cell exhibits very similar kinetic properties to that in the native brush border and to that expressed in Xenopus oocytes. In addition, N-linked glycosylation appears to be important for functional expression of this membrane protein.
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| 4. |
- Björquist, P, et al.
(författare)
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Plasminogen activator inhibitor type-1 interacts exclusively with the proteinase domain of tissue plasminogen activator.
- 1994
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 1209:2, s. 191-202
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Tidskriftsartikel (refereegranskat)abstract
- Two different techniques have been used to study the complex formation of recombinant human plasminogen activator inhibitor type-1, PAI-1, with either recombinant human two-chain tissue plasminogen activator, tc tPA (EC 3.4.21.68), or the tPA deletion variants tc K2P, containing the kringle 2 domain and the proteinase domain, and P, containing only the proteinase domain. The same value for Kon, 2.10(7) M-1s-1 for binding of PAI-1 was found for the three tPA forms by direct detection of the complex formation in real time by surface plasmon resonance, BIAcore, or indirectly by monitoring the time course of the inhibition of tPA using the chromogenic substrate N-methylsulfonyl-D-Phe-Gly-Arg-4-pNA-acetate. Apparently, no conformational change is involved in the rate-limiting step, since the kon value was found to be independent of the temperature from 20 to 35 degrees C. By the BIAcore technique, it was found that the complex between PAI-1 and tPA covalently coupled to the surface, was stable at 25 degrees C, since no dissociation was seen in buffer. However, extended treatment with 1 M NH4OH destroyed the complex with t 1/2 = 5 h. The same kon values and complex composition were found by measuring either the binding of tPA to PAI-1 captured on the monoclonal antibody MAI-11 or the binding of PAI-1 to tPA captured on the monoclonal antibody 2:2 B10. Quantification of the complex composition between PAI-1 captured on the monoclonal antibody MAI-11 with either tPA, K2P or P gave a one-to-one ratio with the fraction of active PAI-1, consistent with the results from SDS-PAGE and the specific activity of PAI-1. The complexes of the three tPA forms with PAI-1 captured on a large surface of MAI-11 dissociated more rapidly from MAI-11, with the same apparent koff, kdis, = 2.10(-3) s-1, compared with 0.7-10(-3) s-1 for the dissociation of PAI-1 alone. In consistance, the Kd, calculated from the direct determination of the kon and koff for the association of different form of PAI-1 to a small surface of MAI-11, was found to be higher for PAI-1 in complex with tPA than for free active PAI-1. Apparently, upon complex formation, a change is induced in PAI-1 at the binding epitope for MAI-11.(ABSTRACT TRUNCATED AT 400 WORDS)
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| 5. |
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| 6. |
- Eriksson, Jan W, et al.
(författare)
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The cGMP-inhibitable phosphodiesterase modulates glucose transport activation by insulin
- 1994
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 1189:2, s. 163-167
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Tidskriftsartikel (refereegranskat)abstract
- To assess the role of the cGMP-inhibitable phosphodiesterase (cGI-PDE) in the action of insulin on glucose transport, adipocytes from young, lean rats were preincubated for 20 min at 37 degrees C with and without OPC 3911, a specific inhibitor of cGI-PDE, and 3-O-methylglucose uptake was measured. Insulin-stimulated glucose transport was impaired by OPC 3911 (approximately 15%) and this impairment became more pronounced in the presence of the degradable cAMP-analogue 8-bromo-cAMP (approximately 45%). This analogue alone did not significantly decrease glucose transport. Furthermore, insulin sensitivity was impaired by the combination of OPC 3911 and 8-bromo-cAMP. Maximal insulin-stimulated glucose transport in adipocytes from aging, obese rats was affected similarly by OPC 3911 and 8-bromo-cAMP, suggesting that cGI-PDE activity is not markedly altered in this insulin-resistant state. In conclusion, cGI-PDE exerts a modulating effect on the stimulatory action of insulin on glucose transport. This effect is particularly pronounced when the cellular cAMP levels are elevated.
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| 7. |
- Gardeström, Per, 1950-
(författare)
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METABOLITE LEVELS IN THE CHLOROPLAST AND EXTRACHLOROPLAST COMPARTMENTS OF BARLEY LEAF PROTOPLASTS DURING THE INITIAL PHASE OF PHOTOSYNTHETIC INDUCTION
- 1993
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0005-2728. ; 1183:2, s. 327-332
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Tidskriftsartikel (refereegranskat)abstract
- Metabolite levels were determined in the chloroplast and extrachloroplast compartments of barley protoplasts during photosynthetic induction using rapid fractionation by membrane filtration. This method allowed studies with a high time resolution the first determination of subcellular metabolite content bring made after only 0.3 s. Upon illumination, dark-adapted protoplasts exhibited a 1 min lag phase prior to commencement of oxygen evolution, and the maximum rate was reached after 4 to 5 min. In contrast to oxygen evolution, the ATP/ADP ratio in the chloroplasts increased from 1 to 2 within 0.5 s and reached a maximum of about 5 after 2 s. There was a dramatic increase in the extrachloroplastic ATP/ADP ratio within a few seconds, reaching a maximum after about 15 s. During the initial phase of photosynthetic induction, the subcellular ATP/ADP ratios were very similar in photorespiratory (low CO,) and non-photorespiratory (high CO,) conditions. The ATP/ADP ratios in both the chloroplast and extrachloroplast compartments remained high until photosynthetic oxygen evolution started and then decreased when the photosynthetic rate reached its maximum. In steady-state photosynthesis the subcellular ATP/ADP ratios were considerably higher under photorespiratory conditions as compared to non-photorespiratory conditions. During the initial phase of photosynthetic induction, 3-phosphoglycerate decreased and triose phosphates increased both in the chloroplast and extrachloroplast compartments. The changes in these metabolites are consistent with a 3-phosphoglycerate/triose phosphate shuttle using the phosphate translocator as the means to supply ATP to the cytosol during photosynthetic induction.
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| 8. |
- Gobl, Anders E, et al.
(författare)
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Menin represses JunD-activated transcription by a histone deacetylase-dependent mechanism
- 1999
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 1447:1, s. 51-56
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Tidskriftsartikel (refereegranskat)abstract
- Recently the multiple endocrine neoplasia type 1 (MEN-1) tumor suppressor gene was cloned. MEN-1 encodes a nuclear protein, called menin, of hitherto unknown function. In order to investigate the biological function of menin we employed the yeast two-hybrid system to identify menin-interacting proteins. Here we report that menin functions as a transcriptional repressor through interaction with the transcription factor JunD. The interaction is mediated via the N-terminal transcription activation domain of JunD, and the C-terminal part of menin. In transient co-transfection experiments, expression of menin leads to specific repression of JunD transcriptional activity, which is dependent on the integrity of the menin C-terminal region. C-Terminal truncations of the protein not only abolish repression, but increase JunD transcriptional activity, implying the existence of a functional domain separate from the JunD-binding region. Menin-mediated repression is relieved by the histone deacetylase inhibitor trichostatin A, indicating that deacetylation of histones is an essential component of this repression mechanism, as has recently been demonstrated for the retinoblastoma protein. Missense, in-frame deletions, frameshift and nonsense mutations lead to inactivation of menin or possibly to truncated proteins. This would result in loss of repression of menin/JunD target genes, as well as non-target genes through indirect mechanisms, deregulation of cellular growth control and endocrine tumorigenesis.
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| 9. |
- Henriksson, Gunnar, et al.
(författare)
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Substrate specificity of cellobiose dehydrogenase from Phanerochaete chrysosporium
- 1998
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 1383:1, s. 48-54
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Tidskriftsartikel (refereegranskat)abstract
- Substrate structural mapping suggests that the catalytic site of cellobiose dehydrogenase from Phanerochaete chrysosporium forms a narrow cave with two hexose binding subsites, Kinetic data also show that beta-di or oligosaccharides are favored electron donors with respect to both KM and kcat. Surprisingly, thiocellobiose showed an even higher kcat than cellobiose, although the KM value was somewhat higher. The CDH was purified using an updated protocol.
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| 10. |
- Hultin, M, et al.
(författare)
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Release of lipoprotein lipase to plasma by triacylglycerol emulsions. Comparison to the effect of heparin.
- 1992
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 1125:1, s. 97-103
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Tidskriftsartikel (refereegranskat)abstract
- It was previously known that lipoprotein lipase (LPL) activity in plasma rises after infusion of a fat emulsion. To explore the mechanism we have compared the release of LPL by emulsion to that by heparin. After bolus injections of a fat emulsion (Intralipid) to rats, plasma LPL activity gradually rose 5-fold to a maximum at 6-8 min. During the same time the concentration of injected triacylglycerols (TG) decreased by about half. Hence, the time-course for plasma LPL activity was quite different from that for plasma TG. The disappearance of injected 125I-labelled bovine LPL from circulation was retarded by emulsion. This effect was more marked 30 min than 3 min after injection of the emulsion. The data indicate that the release of LPL into plasma is not solely due to binding of the lipase to the emulsion particles as such, but involves metabolism of the particles. Emulsion increased the fraction of labelled LPL found in adipose tissue, heart and the red muscle studied, but had no significant effect on the fraction found in liver. The effects of emulsion were quite different from those of heparin, which caused an immediate release of the lipase to plasma, decreased uptake of LPL in most extrahepatic tissues by 60-95%, and increased the fraction taken up in the liver.
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