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Biomedical subjects

A Isaksson

Publications and source records attributed to A Isaksson.

At least 55 records · Page 3Linked to original sources
PubMed1996

Ubiquitin in signal transduction and cell transformation.

Since the discovery of ubiquitin-dependent protein degradation almost two decades ago, great strides have been made towards a detailed understanding of the biochemistry of this process (reviewed in [1-3]). It was, however, only in recent years that the physiological role of the ubiquitin system in signal transduction and the regulation of several cell functions started to be appreciated and experimentally addressed. As with other principal mechanisms of signal transduction, such as phosphorylation or GTP hydrolysis, much of the information regarding the role of the ubiquitin system as a component of cell regulation and signaling cascades, was gained in studies of transformation and the control of cell growth. It seems, however, that ubiquitin-dependent proteolysis, and possibly other processes that are controlled by protein ubiquitination, play a role in many aspects of cellular function from the control of differentiation to intracellular trafficking [1,3,4]. Here we will review some of the results that implicate ubiquitin-dependent proteolysis in the control of cell growth and that indicate how perturbations of ubiquitin-dependent degradation of oncogene and tumor suppressor gene products may contribute to cell transformation and oncogenesis.

Animals↗
PubMed1996

Phosphorylation of Drosophila Jun by the MAP kinase rolled regulates photoreceptor differentiation.

Drosophila Jun (D-Jun) is a nuclear component of the receptor tyrosine kinase/Ras signal transduction pathway which triggers photoreceptor differentiation during eye development. Here we show that D-Jun is a substrate for the ERK-related Drosophila MAP kinase Rolled, which has previously been shown to be a part of this pathway. A D-Jun mutant that carries alanines in place of the Rolled phosphorylation sites acts as a dominant suppressor of photoreceptor cell fate if expressed in the eye imaginal disc. In contrast, a mutant in which the phosphorylation sites are replaced by phosphate-mimetic Asp residues, as well as a VP16-D-Jun fusion protein, can promote photoreceptor differentiation. These data implicate Jun phosphorylation in the choice between neuronal and non-neuronal fate during Drosophila eye development.

Amino Acid Sequence↗
PubMed1996

Plasma beta-hexosaminidase isoenzymes A and B in patients with cerebral infarction.

We determined plasma activity of the isoenzymes of beta-hexosaminidase (Hex) in 151 patients with cerebral infarction, since earlier findings have shown a relation between Hex isoenzymes and risk factors for vascular disease in normal subjects. Compared with 206 control subjects, an elevated level of plasma Hex isoenzymes was found in patients with cerebral infarction, particularly females. However, there was no relation to the clinical subtypes of diagnosis or to the presence of any risk factors for vascular disease, such as carotid artery stenosis, major potential cardio-embolic risk factors on echocardiography, hypertension, heart disease, diabetes mellitus or tobacco smoking. Instead, our findings indicate that Hex isoenzymes in patients with cerebral infarction are more influenced by the level of serum aspartate aminotransferase and blood glucose. The main conclusion is that the liver function as reflected by the level of liver enzymes and glucose metabolism are the major determinants of Hex isoenzymes in plasma.

Aged↗
PubMed1996

beta-Hexosaminidase isoenzymes A and B in middle-aged and elderly subjects: determinants of plasma levels and relation to vascular disease.

Plasma/serum beta-hexosaminidase (Hex) activity is known to be increased in chronic alcoholism, liver disorders, pregnancy and diabetes mellitus. Hex activity also shows an association with risk factors for vascular disease and heredity for arteriosclerosis. There are several isoenzymes of Hex. Using an enzyme immunoassay for Hex isoenzymes (Hex A and Hex B) we studied possible determinants of Hex isoenzymes and their relation to vascular disease in randomly invited (n = 244) 35-95-year-old men and women. In both sexes there were significant age-related increases in Hex activities and men exhibited higher activity of both isoenzymes. Both Hex isoenzymes correlated with age, systolic blood pressure, serum triglycerides and liver enzymes, whereas Hex A was distinguished from Hex B by its stronger correlation with blood glucose. In multiple linear regression analysis Hex A was explained to 20.7% by blood glucose, age, serum aspartate aminotransferase and glutamyl transpeptidase. Hex B was explained to 14% by age, serum glutamyl transpeptidase and serum triglycerides. There was no significant increase in Hex isoenzymes in subjects with hypertension, diabetes mellitus or myocardial disease, nor did current smokers exhibit any increase of these enzymes compared to non-smokers. The main conclusion in that liver function, as reflected by the level of liver enzymes and glucose metabolism, is the major determinant for Hex isoenzymes in plasma.

Adult↗
PubMed1995

Metabolism of homocysteine, its relation to the other cellular thiols and its mechanism of cell damage in a cell culture line (human histiocytic cell line U-937).

This study shows that the intracellular concentration of homocysteine in cultured cells is kept low due to an accumulation in the medium. The intracellular level of homocysteine was decreased when its precursor, methionine, was omitted from the culture medium. Intracellular glutathione and cysteine were lowered in cystine-deficient medium. Intracellular glutathione was also lowered when copper ions were added to the culture medium. It is evident from this study that the intracellular concentration of homocysteine was not influenced by the lowered level of glutathione and/or cysteine. High amounts of homocysteine added to the medium give rise to an increase of intracellular reduced homocysteine, which participates in the transsulfuration pathway and can replace cysteine in the synthesis of glutathione. The addition of relatively high amounts of reduced homocysteine (500 mumol/l) in the presence of copper ions (100 mumol/l) to the culture medium can be directly toxic to the cells, possibly due to oxygen radicals formed by thiol auto-oxidation. Whilst the level of homocysteine in this study using short-time cell culture experiment is much higher than the mild hyperhomocysteinemia thought to be atherogenic in humans, it is conceivable that over a longer time course these levels of homocysteine could be sufficient to induce endothelial dysfunction, eventually leading to atherosclerosis.

Arteriosclerosis↗
PubMed1995

Increases and time-course variations in beta-hexosaminidase isoenzyme B and carbohydrate-deficient transferrin in serum from alcoholics are similar.

beta-Hexosaminidase B-isoforms (beta-hexosaminidase B, P, and intermediate forms; abbreviated herein as "Hex B") and serum carbohydrate-deficient transferrin (CDT) are two markers of alcohol abuse. In the present study, we have compared "Hex B" with CDT as markers of alcohol abuse in a group of alcoholics hospitalized for detoxification after a period of heavy alcohol abuse. We have also followed the disappearance rate of these two markers from circulation. "Hex B" was elevated in 38 of 42 patients hospitalized for detoxication, whereas CDT was elevated in 35 of 42 patients. A highly significant correlation was noted between "Hex B" and CDT in these patients (p = 0.52, p < 0.001). Neither "Hex B" nor CDT correlated with gamma-glutamyltransferase or AST. The disappearance rates from serum of "Hex B" and CDT were determined in 21 hospitalized patients followed for up to 15 days. "Hex B" and CDT showed similar time-course variation and half-lives, 6.5 +/- 3.7 (mean +/- SD) and 8.6 +/- 4.1 days, respectively. The possible reasons for a relation between these two markers are discussed, and it is concluded that more experience of both "Hex B" and CDT in unselected populations is needed to establish the diagnostic potential of these tests as markers of alcohol abuse.

Adult↗
PubMed1995

Liver disease and serum hexosaminidase levels. Studies in a human hepatoma cell-line (Hep G2 cells).

In various forms of liver disease, increased levels of the lysosomal enzyme beta-hexosaminidase (Hex) occur in serum. This may be caused by disturbances of the hepatocytic function, and we therefore studied the intracellular and extracellular isoenzyme pattern of Hex in a human hepatoma cell-line (Hep G2), using an immunoassay method, which separates Hex A and Hex B isoforms. This cell-line synthesizes and secretes Hex. The cumulative release of extracellular activity was about 3-10% of the intracellular activity. B-isoforms comprised one-third of intracellular activity but only 20% of extracellular activity. The proportion of extracellular B-isoforms increased with time, presumably due to instability of A-isoforms at 37 degrees C. Cycloheximide inhibited the release of Hex activity, whereas NH4Cl increased the extracellular fraction of Hex, even at a concentration of 1 mmol/l. We speculate that the increased concentration of NH4+ in patients with liver disease interferes with the distribution pathway of the lysosomal enzymes. This might be one reason for the increased serum Hex activity found in patients with liver disease.

Ammonium Chloride↗
PubMed1995

Beta-hexosaminidase in bile and plasma from patients with cholestasis.

beta-Hexosaminidase (Hex) activity was determined in bile from 18 patients with cholestasis, six patients without cholestasis and in ten normal liver biopsies. The difference in the mean activities in bile from patients with and without cholestasis was not significant. Only about 0.5 promille of total liver Hex activity was lost per day via the bile flow. Gel chromatography showed that enzyme forms present in bile had higher molecular weights than the forms present in liver tissue, indicating that the biliary enzyme was not routed through the lysosomes before release into the bile. In 32 patients with cholestasis, plasma Hex was increased compared to controls, and correlated to bilirubin. The activity was significantly higher in patients with severe cholestasis than in patients with less severe forms of cholestasis, but no significant difference in Hex activity was observed between patients with benign or malignant biliary obstruction. No significant difference was noted between patients with cholestasis for less than 1 week compared to those whose illness had lasted more than 1.5 weeks. The impact of biliary obstruction on plasma Hex is further illustrated by the observation that decompression lowered plasma Hex as well as bilirubin and alkaline phosphatase.

Aged↗
PubMed1995

Plasma beta-hexosaminidase isoenzymes A and B exhibit different relations to blood glucose levels in a population of Type 1 diabetic patients.

The activity of lysosomal enzymes, such as beta-hexosaminidase (Hex), is increased in the plasma and serum of diabetic patients. A positive association has been shown between enzyme activity and glycated proteins, indicating an association with the degree of metabolic control. Several isoenzymes of Hex exist. Studies have reported different proportions of the isoenzymes in plasma from diabetic patients, compared with healthy subjects. In the present study, Hex isoenzymes were examined in 76 Type 1 diabetic patients, of mean age 37.4 years (SD 12.9) compared with 38 age- and sex-matched healthy control subjects in an attempt to evaluate the influence of long- and short-term changes in blood glucose levels on these isoenzymes. The results show that Hex A activity (p<0.01), but not Hex B activity, was higher in the diabetic patients. Hex A activity was positively associated with both the actual blood glucose levels (r = 0.48, p<0.001) and haemoglobin A1c (HbA1c) (r = 0.43, p<0.001), while Hex B activity was associated with the level of HbA1c only (r = 0.42, p<0.001). Both Hex A and B activities were also positively associated with early signs of diabetic nephropathy (e.g. urinary excretion of Hex, fractional albumin excretion ratio and urinary albumin). There was no association between Hex A and B activities and different degrees of retinopathy. In conclusion, the present study demonstrates an association between Hex A and B and metabolic parameters in diabetes as well as with clinical signs of early diabetic nephropathy, but no association with the degree of retinopathy. Furthermore, Hex A seems to be more influenced than Hex B by short-term changes in blood glucose levels.

Adult↗
PubMed1995

Serum beta-hexosaminidase isoenzymes are precursor forms.

The lysosomal enzyme beta-hexosaminidase (Hex, EC 3.2.1.52) was purified from human serum and placental tissue. On gel chromatography, Hex isoenzymes (Hex A, B and P) from serum showed a molecular weight of about 150 kDa, i.e. higher than the 120 kDa found for the placental (tissue) isoenzymes (Hex A and B). Sodium dodecyl sulphate polyacrylamide gel electrophoresis of non-reduced serum Hex A revealed two different subunits, denoted alpha and beta, corresponding to apparent molecular weights of 70 and 67 kDa, respectively. Serum Hex B and Hex P were composed of beta-subunits only. In contrast, the apparent molecular weights of the alpha- and beta-subunits originating from tissue were both 55 kDa. Reduction of tissue Hex A and Hex B resulted in cleavage of the beta-subunit into several fragments, whereas the beta-subunit in serum forms was resistant to this treatment. These results are consistent with the serum forms never having entered the lysosome prior to their release to the extracellular environment and serum forms can therefore be classified as precursor molecules. No mature (tissue) forms were found in serum. Isoelectric focusing before and after neuraminidase treatment of the serum isoenzymes showed that they all contained sialic acid and that Hex B and Hex P had an identical focusing pattern after desialylation. Serum beta-hexosaminidase is frequently elevated in liver disease, alcoholism and pregnancy and the results of the present study indicate that this is due to an increased synthesis and secretion of hypersialylated beta-subunits, rather than to release of intralysosomally stored enzyme.

Antibodies, Monoclonal↗
PubMed1994

Intraoperative monitoring of intact PTH during surgery for primary hyperparathyroidism.

The intraoperative differential diagnosis between adenoma and hyperplasia during surgery for primary hyperparathyroidism (pHPT) is sometimes difficult. Several methods have been proposed to aid the surgeon in deciding on the amount of parathyroid tissue to be resected. We examined the use of intraoperative monitoring of intact PTH in 47 patients operated upon for pHPT. The highly sensitive assay for intact PTH was modified to permit a total turn-around time from gland excision to obtained result of about 60 min. The correlation (r) between the results of the modified and the conventional method, which requires 24 h of incubation, was 0.98. At 15 min after removal of the parathyroid adenoma the levels of intact PTH had decreased by [mean (SD)] 85 (11)%. A decrease of 63% in intact PTH in patients with parathyroid adenoma predicted with 95% confidence the 4 patients with primary hyperplasia as not having parathyroid adenoma. We conclude that intraoperative measurement of intact PTH could be a valuable adjunct to surgical skill, especially for reoperative parathyroid surgery.

Adenoma↗
PubMed1994

Serum beta-hexosaminidase isoenzymes are related to risk factors for atherosclerosis in a large population of postmenopausal women.

Lysosomes are involved in atherogenesis. Therefore we have studied the level of serum beta-hexosaminidase isoenzymes (Hex A and Hex B) in relation to risk factors for atherosclerosis in a homogeneous population of 886 post-menopausal women. We found a relation with several risk factors such as serum triglycerides, diastolic and systolic blood pressure, blood glucose, waist/hip ratio and body mass index but not with serum cholesterol. Also, the mean values for Hex A and Hex B were higher in smokers than in non-smokers but only the mean value for Hex A differed significantly. The relation of serum beta-hexosaminidase isoenzymes to risk factors might be due to lysosomal over-loading, which gives rise to increased enzyme synthesis and enhanced secretion of lysosomal enzymes to circulation. The subjects in the 95-100 percentile of Hex A showed significantly increased frequency of myocardial infarction of their fathers and of stroke in their mothers and the subjects in the 95-100 percentile of Hex B showed increased frequency of stroke in their mothers. Thus the findings of a relation between Hex isoenzymes and heredity for vascular disease further stress the significant relation between Hex isoenzymes and risk factors. Since Hex B is a sensitive marker for alcohol abuse, we also investigated its serum level in subjects that could be suspected of alcohol abuse. However, we did not find any differences in these subjects compared with the others, possibly due to the relatively short half-life of Hex B after alcohol withdrawal.

Alcoholism↗
PubMed1994

Lysosomes and human liver disease: a biochemical and immunohistochemical study of beta-hexosaminidase.

Liver biopsies from 88 patients with different liver diseases were studied for beta-hexosaminidase activity. Liver specimens with normal light microscopical morphology showed no immunohistochemical reactivity for beta-hexosaminidase. Increased reactions were noted, mainly in hepatocytes, in biopsies from the patients with different liver diseases. A very large interindividual variation of biochemical liver beta-hexosaminidase activity occurred even within the same diagnostic group, and no group of patients showed any significant increase of liver beta-hexosaminidase activity compared with the patients with normal liver histology. Livers with positive immunohistochemistry showed increased biochemical values for beta-hexosaminidase. In patients with cholestasis due to alcohol abuse, the immunohistochemical reaction was intense and the biochemical beta-hexosaminidase activity was significantly increased compared with non-alcoholic cholestatic cases. Furthermore, blood samples taken from 50 patients at the same time as the liver biopsies. These patients showed significantly increased serum beta-hexosaminidase activity compared with normal controls, but no correlation was found between beta-hexosaminidase activities in the liver and serum of these patients.

Adolescent↗
PubMed1993

Release of beta-hexosaminidase isoenzymes in cultured human fibroblasts.

We have used enzyme immunoassay methods to study the intra- and extracellular isoenzyme pattern of beta-hexosaminidase (Hex) in human fibroblast cultures. The released activity into the medium during 24 h was about 10% of the intracellular activity. Intra- and extracellular ratio of Hex B to total Hex (Hex A plus Hex B) was about one-third. Estimation of the molecular weight of the released activity showed that it corresponded to 150 kDa, which is equal to the high molecular mass precursor forms of the enzyme. NH4Cl is known to disturb the intracellular transport of lysosomal enzymes and increase the secretion of newly synthesized precursor forms. Addition of NH4Cl even at 3 mM resulted in an increased release of total Hex, which was already noted within 24 h. We speculate that the increased concentration of plasma NH4+ in patients with liver disease interferes with the intracellular distribution pathway of the lysosomal enzymes and this could contribute to the increased content of lysosomal enzymes present in plasma from these patients. Tunicamycin (0.3-3.0 micrograms/l) increased the percentage of Hex B in the medium, whereas an increased release of Hex was noted only after 48 h. Tunicamycin is known to enhance the secretion of N-linked oligosaccharide-free forms of lysosomal enzymes. Thus the oligosaccharide chains on alpha- and beta-subunits seem to be important for the normal formation process of the Hex A isoenzyme.

Ammonium Chloride↗
PubMed1993

L-iduronate-rich glycosaminoglycans inhibit growth of normal fibroblasts independently of serum or added growth factors.

The effects of various glycosaminoglycans (GAGs) on the growth rate of normal fibroblasts and a fibrosarcoma cell line (HT 1080) were examined. Cells were grown in 96-well microplates in the absence or presence of serum mitogens, epidermal (EGF), platelet-derived (PDGF), acidic fibroblast (aFGF), or basic fibroblast growth factor (bFGF). Cell number was measured by using crystal violet to stain cell nuclei (Westergren-Thorsson, G., Onnervik, P.-O., Fransson, L.-A., and Malmström, A. J. Cell. Phys. 147, 523-530, 1991) and also by using a Coulter counter. In the presence of serum mitogens, L-iduronate (IdoA)-rich GAGs, such as dermatan sulfate, heparin, and highly sulfated heparan sulfate, inhibited proliferation of normal cells (25-35%), whereas HT 1080 cells were unaffected or slightly stimulated. Ham's F-12 supplemented with insulin and transferrin but without growth factors was able to support growth of both cell types. Under these conditions, the IdoA-rich GAGs still suppressed growth of normal cells (40-55%), whereas HT 1080 cells again responded poorly. When growth factors were added proliferation of normal fibroblasts was further stimulated, EGF being the most effective. In the presence of either EGF, PDGF, or bFGF, IdoA-rich GAGs had a sustained inhibitory effect on normal fibroblasts (30-50% at concentrations at or above 10 micrograms/ml). However, in the presence of aFGF, both IdoA-rich and IdoA-poor heparan sulfates enhanced growth (nearly twofold after prolonged exposure) suggesting a stabilization of this growth factor. In general, IdoA-rich GAGs appear to inhibit proliferation of normal cells irrespective of the type of growth factor used. Therefore, GAGs are likely to act directly on cell-derived regulatory components, either before or after internalization. As fibrosarcoma cells were much less sensitive to growth inhibition, they may contain altered receptors for GAGs.

Blood Proteins↗