1.
Abrahamson, Magnus
(author)
Molecular basis for amyloidosis related to hereditary brain hemorrhage
1996
In: Scandinavian journal of clinical and laboratory investigation. Supplementum. - 0085-591X. ; 56:226, s. 47-56
Journal article (peer-reviewed) abstract
The aim of the project has been to elucidate molecular events leading to amyloidosis in Hereditary Cystatin C Amyloid Angiopathy (HCCAA) patients, to enable simple diagnosis of the disease and with the ultimate goal to understand the amyloid formation process in detail, in order to develop inhibitors to the process. At the DNA level, a point mutation segregating with HCCAA was identified in the cystatin C gene on chromosome 20, after basic characterization of cDNA and gene for the wildtype protein. The mutation results in the amino acid substitution Leu-68-Gin (L68Q) and abolishes a recognition site for Alu I. This information was used to design a PCR based assay for simple and rapid mutation detection in DNA from blood samples to allow routine diagnosis of HCCAA. Studies at the protein level, allowed through E. coli expression of wildtype and L68Q mutated cystatin C genes, revealed that both protein variants effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation: 0.4 and 0.3 nM, respectively), but differ considerably in their tendency to dimerize and form aggregates. The initial dimerization of L68Q-cystatin C results in complete loss of biological activity and is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. This result might be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates. The three-dimensional structure of normal cystatin C, crystallized in a complex with cathepsin B, was elucidated by X-ray analysis and subsequent refinement of the structure to 3.0 A resolution. Besides pinpointing the cystatin C structures resulting in efficient target enzyme inhibition, the results demonstrated that the Leu-68 residue is buried in the hydrophobic core of the protein. Studies of the three-dimensional solution structure of wildtype cystatin C by NMR spectroscopy revealed that cystatin C dimers can be formed as a result of slight, localized structural changes under conditions preceding complete defolding and denaturation of the protein. Dimers of L68Q-cystatin C are likely similar but are formed at temperatures nearly 30 degrees C lower than needed for the wildtype protein, indicating that the Leu-68-Gln substitution lowers the transition temperature for unfolding. Thus, the results presented suggest that cystatin C provides a system where decreased stability of a mutant protein correlates with its amyloidogenic nature. The NMR results furthermore imply that the hydrophobic proteinase-binding region of cystatin C is directly involved in dimer formation and that compounds designed to interact with this region could serve as inhibitors to the dimerization, and likely also the subsequent amyloid formation process, of cystatin C in HCCAA patients.
2.
Agardh, Carl-David, et al.
(author)
Glucose levels and insulin secretion during a 75 g glucose challenge test in normal pregnancy
1996
In: Journal of Internal Medicine. - : Wiley. - 1365-2796 .- 0954-6820. ; 240:5, s. 303-309
Journal article (peer-reviewed) abstract
OBJECTIVES: The aim of the study was to evaluate glucose levels and insulin secretion early in pregnancy and at a time when gestational diabetes mellitus frequently occurs in order to define reference values for glucose tolerance during pregnancy. The results were also related to maternal factors that might identify subjects at risk of developing gestational diabetes mellitus as well as foetal factors that might be a result of impaired glucose tolerance during pregnancy. DESIGN: A prospective study. SETTING: All Caucasian women attending one antenatal out-patient care unit were offered a 75 g oral glucose tolerance test at the 17th and 32nd week of gestation. SUBJECTS: A total of 586 consecutive pregnant women were included in the study. All 586 women were examined by repeated blood glucose measurements and 298 agreed to perform oral glucose tolerance tests as well. MAIN OUTCOME MEASURES: Venous whole blood glucose values were measured in the fasting state and in samples obtained 15, 30, 45, 60, 75, 90 and 120 min after oral intake of 75 g glucose. Serum insulin and C-peptide were also measured at these times. In all subjects, a random blood glucose sample was taken at the first visit, and thereafter at the 20th, 30th and 36th week of gestation. Information was also obtained from all subjects regarding body mass index, weight gain during pregnancy, smoking habits, family history of diabetes and hypertension, hypertension during pregnancy, past obstetric history, parity, and fetal outcome. RESULTS: The glucose tolerance was significantly impaired at the 32nd week of gestation compared with the 17th week of gestation. The mean +2SD 2 h glucose value during the oral glucose tolerance test at the 32nd week of gestation was 8.0 mmol L-1. Impaired glucose tolerance was characterised by increased insulin resistance, with a significant rise in serum insulin and C-peptide concentrations and in the insulin/glucose index during the oral glucose tolerance test at the 32nd week of gestation. Maternal factors associated with an impaired glucose tolerance were a family history of diabetes mellitus, smoking, a weight gain more than 18 kg during pregnancy, and glucosuria, while a family history of hypertension and hypertension present during pregnancy were not. Foetal factors that might be a result of impaired glucose tolerance during pregnancy, e.g. macrosomia and prematurity as well as complicated deliveries such as vacuum extraction/forceps or Caesarean section, all tended to be associated with higher blood glucose values. The same pattern was seen when the Apgar score was < 7. CONCLUSIONS: The results from this study show that the present cut-off values for diagnosis of gestational diabetes mellitus should be revised. Even if some maternal factors might indicate an increased risk for impaired glucose tolerance during pregnancy, they are probably not enough to detect women with gestational diabetes mellitus. Therefore, a screening programme for gestational diabetes should be considered.
3.
Arnadottir, M, et al.
(author)
The effect of reduced glomerular filtration rate on plasma total homocysteine concentration
1996
In: Scandinavian Journal of Clinical & Laboratory Investigation. - : Informa UK Limited. - 1502-7686 .- 0036-5513. ; 56:1, s. 41-46
Journal article (peer-reviewed) abstract
The concentration of homocysteine in plasma has been shown to be increased in renal failure, possibly contributing to the accelerated atherosclerosis observed in uraemic patients. The aim of the present study was to document the relationship between plasma total homocysteine (tHcy) concentrations and glomerular filtration rates (GFR) in highly selected patients, with renal function ranging from normal to dialysis dependency. GFR was defined as the plasma clearance of iohexol; a more accurate method than the creatinine-based estimations applied in previous studies. Plasma tHcy concentrations were highly correlated to GFR (r = -0.70, p < 0.0001) and were significantly increased already in moderate renal failure. According to a multiple regression analysis, GFR and red cell folate concentrations independently predicted plasma tHcy concentrations, whereas those of serum creatinine, plasma pyridoxal-5-phosphate, urine albumin and urine alpha-1-microglobulin (a marker of tubular damage) did not. Thus, GFR seems to be a better determinant of plasma tHcy concentration than serum creatinine concentration. Plasma total cysteine and total cysteinylglycine concentrations followed the same pattern as those of tHcy.
4.
Bjork, Ingemar, et al.
(author)
The importance of the second hairpin loop of cystatin C for proteinase binding. Characterization of the interaction of Trp-106 variants of the inhibitor with cysteine proteinases
1996
In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 35:33, s. 10720-10726
Journal article (peer-reviewed) abstract
The single Trp of human cystatin C, Trp-106, is located in the second hairpin loop of the proteinase binding surface. Substitution of this residue by Gly markedly altered the spectroscopic changes accompanying papain binding and reduced the affinity for papain, actinidin, and cathepsins B and H by 300-900-fold. The decrease in affinity indicated that the side chain of Trp-106 contributes a similar free energy, -14 to -17 kJ·mol-1, to the binding to all four cysteine proteinases, corresponding to about 20-30% of the total binding energy. Replacement of Trp-106 by Phe led to a smaller (30-120-fold) decrease in affinity for the four enzymes than Gly substitution. The binding energy of the Phe residue corresponded to 20-45% of that of Trp, showing that a phenyl group can only partly substitute for the indole ring. The reduced affinities of the cystatin C Trp-106 variants for all proteinases studied were due almost exclusively to increased dissociation rate constants. The second hairpin loop thus contributes to the binding primarily by keeping cystatin C anchored to the proteinase once the complex has been formed. This role is partly in contrast to that of the N-terminal region, which increases the affinity of cystatin C for cathepsin B by increasing the association rate constant. Removal of the N-terminal region of the Trp-106Gly variant by proteolytic cleavage substantially weakened the binding to papain and cathepsin B. The resulting affinity indicated that the first hairpin loop (the "QVVAG-region"), which is the only region of the proteinase binding surface remaining intact in the truncated variant, contributes 40-60% of the total free energy of binding of cystatin C to both proteinases.
5.
Ekbom, Tord, et al.
(author)
Decrease in high density lipoprotein cholesterol during prolonged storage. CELL Study Group
1996
In: Scandinavian Journal of Clinical & Laboratory Investigation. - : Informa UK Limited. - 1502-7686 .- 0036-5513. ; 56:2, s. 97-101
Journal article (peer-reviewed) abstract
Different studies on the stability of high density lipoprotein cholesterol (HDL) in frozen serum or plasma have yielded conflicting results, namely increase, decrease, or no change at all during prolonged storage under freezing conditions. As part of a major trial on lipid-lowering strategies we statistically demonstrated a time-related decrease in HDL cholesterol during storage up to 46 months at -20 degrees C. We therefore re-analysed 85 frozen samples that had been analysed fresh and then stored from 26 to 46 months, using the dextran sulphate 500/Mg2+ method. A linear regression analysis of change in HDL cholesterol on time was performed. The slope was significantly negative (p < 0.0005). The regression equation was (decrease in HDL) = 0.05 - 0.008 x (time in months), i.e. after 6 months' storage at -20 degrees C there was almost a 1% decrease in the HDL cholesterol concentration per month of storage.
6.
Ekiel, Irena, et al.
(author)
Folding related dimerization of human cystatin C
1996
In: Journal of Biological Chemistry. - 1083-351X. ; 271:3, s. 1314-1321
Journal article (peer-reviewed) abstract
With the aim to improve our understanding of the structural basis for protein self-association and aggregation, in particular in relationship to protein refolding and amyloid formation, folding-related processes for human cystatin C have been studied. Using NMR spectroscopy together with chromatographic and electrophoretic methods, a self-association process resulting in dimer formation for protein samples treated with denaturing agents as well as for samples subjected to low pH or high temperature conditions could be studied with amino acid resolution. In all three cases, the dimerization involves properly folded molecules and proceeds via the reactive site of the inhibitor, which leads to complete loss of its biological activity. This dimerization process has potential relevance for amyloid formation by the brain hemorrhage-causing Leu-Gln variant of cystatin C. The results also indicate that cystatin C dimerization and inactivation may occur in acidified compartments in vivo, which could be relevant for the physiological regulation of cysteine proteinase activity.
7.
Håkansson, Katarina, et al.
(author)
Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution
1996
In: Comparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology. - : Elsevier BV. - 1879-1107 .- 1096-4959. ; 114:3, s. 303-311
Journal article (peer-reviewed) abstract
Recombinant mouse (Mus musculus) and rat (Rattus norvegicus) cystatin C were produced by expression in Escherichia coli, isolated and functionally characterized. The mouse and rat inhibitors were both fully active in titrations of papain. Determination of equilibrium constants for dissociation (Ki) for their complexes with the target proteinase, cathepsin B, produced values not largely different from that for human cystatin C (Ki 0.07-0.13 nM). Rabbit antisera against mouse and rat cystatin C were produced and used for improved affinity purification of the recombinant inhibitors. Affinity purified immunoglobulins isolated from the antiserum against mouse cystatin C were used for construction of a sensitive enzyme-linked immunosorbent assay. The assay was used to demonstrate a high degree of immunological cross-reactivity between mouse and rat cystatin C and could be used for cystatin C quantification in mouse and rat tissue homogenates. All tissues analyzed contained cystatin C, with a relative content very similar to that of human tissues. For all species, brain tissue contained the highest cystatin C amounts and liver the lowest, whereas kidney, spleen and muscle tissues were intermediate in content. In the mouse, a notable high cystatin C content in parotid gland tissue was observed. The high degree of similarity in distribution pattern and functional properties for mouse, rat and human cystatin C indicates that a murine model should be relevant for studies of the human disease, hereditary cystatin C amyloid angiopathy.
8.
Larsson, Jan, et al.
(author)
Effects of TRH and atropine on induction and duration of anesthesia with propofol in rats.
1996
In: Peptides. - : Elsevier BV. - 0196-9781 .- 1873-5169. ; 17:2, s. 293-297
Journal article (peer-reviewed) abstract
The effects of IV TRH pretreatment on induction of anesthesia with propofol or pentobarbital were investigated in rats. The effects of IV TRH, administered after induction, on duration of propofol anesthesia and the interaction with atropine were also studied. The doses of propofol or pentobarbital were not influenced by TRH. TRH reduced duration of anesthesia after propofol, with higher brain concentrations of propofol at recovery. Atropine did not block this effect, but given alone prolonged duration of anesthesia. It is concluded that TRH shortens the duration of propofol anesthesia, probably due to a pharmacodynamic effect and not to a pharmacokinetic interaction.
9.
Lindeberg, Staffan, et al.
(author)
Lipoprotein composition and serum cholesterol ester fatty acids in nonwesternized Melanesians
1996
In: Lipids. - 0024-4201. ; 31:2, s. 153-158
Journal article (peer-reviewed) abstract
In this study, the relationships between dietary fat [as measured by serum cholesterol ester fatty acids (CE-FA)], age, smoking, body mass index, and serum lipids were analyzed in 151 subsistence horticulturalists, aged 20-86 yr, from Kitava, Trobriand Islands, Papua New Guinea. Their diet consists of tubers, fruit, coconut, fish, and vegetables with a negligible influence of western food and alcohol. Total fat intake is low [21% of energy (en%)], while saturated fat intake from coconuts is high (17 en%, mainly lauric and myristic acid). In multivariate analysis, 11-43% of the variation of the serum lipoprotein composition was explained by CE-FA, age, and smoking habits. The proportion of CE20:5n-3 explained much of the variation of triglycerides (TG, negative relation) and high density lipoprotein-cholesterol (HDL-C, positive) in both sexes and serum apolipoprotein A1 (ApoA1, positive) in the males. CE16:0 was positively related to TG and negatively related to HDL-C and ApoA1 in both sexes, and in males it related negatively to total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C). In males, negative relationships were present between CE18:2n-6 and TC and between CE14:0 and serum lipoprotein(a). Smoking was independently associated with lower ApoA1 in both sexes and with lower HDL-C and higher TG, TC, LDL-C, and apolipoprotein B in males. In conclusion, marine n-3 fatty acids and linoleic acid showed the same potentially beneficial relationships with lipoproteins and apolipoproteins as in western populations. The relations of palmitic acid to serum lipids may be explained in terms of endogenous fat synthesis at a low-fat intake, rather than reflecting its relative intake.
10.
Magnusson, Beatrice M., et al.
(author)
Effects of topical application of capsaicin to human skin : a comparison of effects evaluated by visual assessment, sensation registration, skin blood flow and cutaneous impedance measurements.
1996
In: Acta Dermato-Venereologica. - 0001-5555 .- 1651-2057. ; 76:2, s. 129-132
Journal article (peer-reviewed) abstract
A new non-invasive device, which enables local measurements of electrical impedance, has been used to evaluate the degree of irritation in human skin. The results have been compared with visual scoring, sensations and laser Doppler flowmetry. Capsaicin (50 microliters 1% solution) and control solutions (50 microliters 50% ethanol) were applied in a chamber for 30 min on the volar forearm of 7 volunteers. Values were recorded before application and during the total test period of 4.5 h. Sensations like sting/prick, burn and pain were produced by this treatment, and the flare response was observed. Using the non-invasive laser Doppler flow technique to measure blood flow in human skin, we have shown that topical application of capsaicin abolishes the vasodilator response to local heat provocation (40 degrees C). There was close agreement among values obtained using visual assessments, sensations and laser Doppler flowmetry. Results obtained using electrical impedance measurements were not consistent with the other three methods.
