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Biomedical subjects

A Laine

Publications and source records attributed to A Laine.

At least 19 recordsLinked to original sources
PubMed1992

Variations in the glycoforms of serum alpha 1-antichymotrypsin in liver diseases and after liver transplantation.

1. Using crossed immunoaffinity electrophoresis with free concanavalin A in the first dimension, we studied the microheterogeneity of alpha 1-antichymotrypsin due to various glycoforms in sera from patients with various liver diseases and after liver transplantation. 2. Studies by isoelectric focusing and immunoblotting and by crossed immunoelectrophoresis without concanavalin A in the first dimension allowed us to show that there is no dramatic variation in electrophoretic heterogeneity of alpha 1-antichymotrypsin in the serum of patients with liver diseases or after liver transplantation when compared with that of normal subjects. Therefore the heterogeneity observed in crossed immunoaffinity electrophoresis is due to various interactions with concanavalin A. 3. The results were expressed as the ratio of concanavalin A non-reactive glycoforms plus concanavalin A weakly reactive glycoforms to concanavalin A reactive glycoforms, called R alpha 1-ACT. R alpha 1-ACT was significantly higher in patients with cirrhosis (n = 53) when compared with normal subjects (n = 30). The median R alpha 1-ACT was 1 (range 0.72-1.25) in normal subjects. It was 1.6 (range 1.18-3.02), 1.45 (range 0.65-4.12) and 2.24 (range 1.03-19) in cirrhosis of Child's grade A, B and C, respectively. There was a dramatic decrease in glycoforms with mostly biantennary glycans in some patients with Child's grade C cirrhosis. Serum levels of alpha 1-antichymotrypsin were lower than normal only in some patients with Child's grade C cirrhosis. 4. Among the patients with acute viral hepatitis studied (n = 17), five were studied longitudinally.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Structure determination of the glycans of human-serum alpha 1-antichymotrypsin using 1H-NMR spectroscopy and deglycosylation by N-glycanase.

alpha 1-Antichymotrypsin purified from normal human serum was separated by affinity chromatography into th ree microheterogeneous forms on a concanavalin-A-Sepharose column: a pass-through (peak 1), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine-linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re-N-acetylation and further separated by affinity chromatography on a concanavalin-A-Sepharose column. The complete primary structure of the glycans was determined by high-resolution 1H-NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N-glycanase was used for the deglycosylation of the unfractionated alpha 1-antichymotrypsin; the successive removal of the N-linked complex-type oligosaccharide side chains of alpha 1-antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that alpha 1-antichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak 1 contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.

Amidohydrolases↗
PubMed1991

Effects of tumour mass and circulating antigen on the biodistribution of 111In-labelled F(ab')2 fragments of human prostatic acid phosphatase monoclonal antibody in nude mice bearing PC-82 human prostatic tumour xenografts.

We have evaluated the effects of tumour mass and circulating antigen (prostatic acid phosphatase, PAP) on the biodistribution and the incorporation of 111In-labelled F(ab')2 monoclonal antibody (MoAb) fragments directed against human PAP into human prostatic tumours (PC-82; 0.1-8.9 g) growing in nude mice. The radioactivities in the blood, liver, spleen, kidney and tumour were compared at 1, 3, 4 and 6 days after the intravenous administration of the antibody fragments. There was a significant correlation between the tumour size and the serum PAP concentration in the model employed. Even tissue of a small tumour (less than 0.1 g) had a high concentration of PAP, but it was not secreted into the circulation in detectable amounts when measured by radioimmunoassay (the lowest standard was 0.5 micrograms/l). The percentage uptake by tumours of the injected dose per gram of tissue (%ID/g) was inversely proportional to the tumour size at 24 h after the administration of 111In-labelled F(ab')2 fragments. This relationship had levelled off by 72 h and most likely reflected a better vascularisation of the smaller tumours. Our results show that the increase in tumour size and in the concentration of circulating antigen in the blood led to decreased tumour-to-blood ratios, since there was a tendency for higher blood activities in mice with larger tumours and higher serum PAP concentrations. There was no correlation between tumour size and label uptake by the liver during the follow-up over 144 h, although serum PAP concentrations ranged from 3.1 micrograms/l to 352 micrograms/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1991

Deglycosylation of alpha 1-proteinase inhibitor is impaired in the faeces of patients with active inflammatory bowel disease (Crohn's disease).

1. alpha 1-Proteinase inhibitor (alpha 1-antitrypsin) was excreted in the faeces of patients with inflammatory bowel disease in different molecular forms: Mr-51,000 and Mr-45,000 forms were widely found in the stools of patients with active disease, whereas a Mr-38,000 species was frequently recovered from healthy subjects and patients with quiescent disease (Mizon, Becuwe, Balduyck et al. Clin. Chem. 1988; 34, 2268-70). 2. N-Terminal sequencing of the Mr-38,000 form of alpha 1-proteinase inhibitor, after SDS/PAGE and electrotransfer on polyvinyl difluoride membranes, showed that it differed from plasma alpha 1-proteinase inhibitor by the loss of 17 N-terminal amino acids. 3. Carbohydrate analysis of the isolated Mr-38,000 form revealed a total lack of neutral sugars. 4. In contrast, the Mr-51,000 form of alpha 1-proteinase inhibitor is glycosylated and thus could be differentiated by virtue of its reactivity with concanavalin A. The analysis of 25 faecal extracts from patients with Crohn's disease allowed us to confirm that the presence of the glycosylated form of alpha 1-proteinase inhibitor was closely related to the degree of inflammation. 5. From these data, it may be hypothesized that the hydrolytic activity of some glycosidases is greatly reduced in active Crohn's disease.

Adolescent↗
PubMed1991

[Glycoforms of serum alpha 1-antichymotrypsin studied by immunoaffino-electrophoresis. From the fundamental aspect to clinical applications].

alpha 1-antichymotrypsin, purified or in whole serum, exhibits microheterogeneous forms when studied by crossed immunoaffinoelectrophoresis with free concanavalin A (Con A) in the first dimension. alpha 1-antichymotrypsin purified from the serum of a single healthy donor was separated into three forms by affinity chromatography on a Con A-Sepharose 4B column: a Con A non-reactive form, a Con A weakly reactive form and a Con A reactive form. Some of their physico-chemical properties are compared. The complete primary structure of the glycans of each form was determined by high resolution 1H-NMR spectroscopy. The results indicated the presence of diantennary and triantennary type glycanic structures which occur frequently in serum glycoproteins. From deglycosylation experiments it is concluded that alpha 1-antichymotrypsin carries four oligosaccharide side chains. The Con A non-reactive form contains four triantennary glycans, the weakly reactive form contains three triantennary and one diantennary glycans and the Con A reactive form possesses on average one triantennary and three diantennary glycans. Significant variations in the relative ratios of the microheterogeneous forms were detected in various inflammatory syndromes. There is an increased proportion of Con A non-reactive form in patients developing a systemic disease (systemic lupus erythematosus, rheumatoid arthritis, temporal arteritis). In contrast, the proportion of Con A non-reactive form decreased in patients with acute septic inflammation whereas no variation appeared in patients with metastatic breast cancer. We also studied the variations of alpha 1-antichymotrypsin microheterogeneity in sera from patients with giant-cell arteritis and/or polymyalgia rheumatica before and during treatment with glucocorticoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1990

Comparison of the effects of purified human alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor on NK cytotoxicity: only alpha 1-proteinase inhibitor inhibits natural killing.

While an inhibitory effect on natural killer (NK) cell activity was demonstrated with partially purified alpha 1 Achy, neither highly purified alpha 1 Achy from two healthy donors nor from one patient with giant-cell arteritis, which carries more highly branched glycans, inhibited the NK cytotoxicity. Our purification procedure, based on immunoaffinity chromatography and gel filtration, was not in question since the pure alpha 1-proteinase inhibitor (alpha 1PI) prepared in our laboratory by using a similar procedure continued to inhibit the NK cytotoxicity. If an inhibitory effect not related to antiprotease activity occurs with alpha 1PI, it is surprising that it is not shared by alpha 1 Achy which, like alpha 1PI, belongs to the serpin family and which possesses a strong structural homology with alpha 1PI. Our finding that alpha 1PI is able to affect human NK cytotoxicity while alpha 1 Achy (even with more highly branched glycans) is unable to suggests that events controlling NK activity may involve other enzymes than chymotrypsin-like enzymes.

Arteritis↗
PubMed1990

Microheterogeneity of alpha 1-antichymotrypsin in the management of giant-cell arteritis and polymyalgia rheumatica.

1. Using crossed immunoaffinity electrophoresis with free concanavalin A in the first dimension, we studied the glycan microheterogeneity of alpha 1-antichymotrypsin in sera from patients with giant-cell arteritis and/or polymyalgia rheumatica, and its variation in the serum of several of these patients during induction of disease remission by prednisone therapy and possible further recurrence of giant-cell arteritis and/or polymyalgia rheumatica. 2. In the serum of patients with active disease we observed increased proportions of concanavalin A nonreactive and concanavalin A weakly reactive fractions. The results were expressed as the ratio of concanavalin A non-reactive fraction plus concanavalin A weakly reactive fraction to concanavalin A reactive fraction, called R alpha 1-ACT. An R alpha 1-ACT higher than 1.8 (upper normal value) was found in 30/31 sera from patients with active disease (sensitivity 97%) and in 2/22 sera from patients with inactive disease (specificity 91%). 3. The erythrocyte sedimentation rate and the serum C-reactive protein level, common markers of biological inflammation, are usually elevated in active giant-cell arteritis and/or polymyalgia rheumatica. The two parameters are commonly used to guide the therapy during the course of the disease, but they have no specificity. An erythrocyte sedimentation rate of greater than 30 mm/h was found in 30/31 sera from patients with active disease (sensitivity 97%) and in 5/22 sera from patients with inactive disease (specificity 77%).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1989

The microheterogeneity of serum alpha 1-antichymotrypsin revealed by interaction with concanavalin A in crossed immunoaffinoelectrophoresis and in affinity chromatography.

In crossed immunoaffinoelectrophoresis with free concanavalin A in the first dimension, human serum alpha 1-antichymotrypsin, purified or in whole serum, exhibited four peaks in presence of 0.02 M alpha-methyl-D-glucoside added to the second-dimensional gel. alpha 1-Antichymotrypsin purified from the serum of a single healthy donor was separated by affinity chromatography into three fractions on a laboratory-prepared concanavalin A-Sepharose 4B column: a pass-through fraction, a retarded fraction and a bound fraction, eluted from the column on addition of sugar to the buffer. These three fractions were analyzed by crossed immunoaffinoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing before and after desialylation. The results of electrophoresis as well as chemical analyses indicate that these microheterogeneous forms carry glycans with decreasing degrees of branching from the concanavalin A-pass-through form to the concanavalin A-bound form. This approach represents a first step towards the elucidation of the molecular basis of the microheterogeneity of alpha 1-antichymotrypsin.

Amino Acids↗
PubMed1989

Changes induced by short-term xylene exposure in human evoked potentials.

Nine healthy male volunteers were exposed to m-xylene for 3 h in the morning and 40 min in the afternoon with a 40-min break in between. The atmospheric m-xylene concentrations were either stable at 8.2 mumol/l (200 ppm) or they fluctuated (5.2-16.4 mumol/l; 135-400 ppm) with peaks of 16.4 mumol/l and duration of 20 min at the beginning of each exposure session. The subjects were either sedentary or exercised at 100 W for 10 min at the beginning of each session during both exposure types. The two control days, with and without exercise, were similar to the exposure days but without exposure. Evoked potentials were recorded in the morning before the exposure and immediately after the morning and afternoon sessions. Visual evoked potentials were studied to a pattern reversal stimulus (pattern VEP) and to a light flash (flash VEP). For pattern VEPs the latencies of P50, N70, P100, N135 and P170 as well as the peak-to-peak amplitude of N70 to P100 were measured. For flash VEPs the latencies of P50, N70, P100, N150 and P200 as well as the peak-to-peak amplitude of P100 to N150 were measured. Short-latency auditory evoked potentials arising in the brainstem (BAEP) were recorded for a click stimulus. The peaks I, II, III, IV and V were identified from the grand averages. The effect of various exposure paradigms was evaluated by comparing the individual changes on an exposure day to those during the control days.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Qualitative and quantitative comparison of brain proteins in Alzheimer's disease.

In human brain extracts, most proteins of pathological interest in Alzheimer's disease are insoluble and their analysis is often performed on denatured and reduced samples by immunoblotting after electrophoresis on polyacrylamide gel in presence of sodium dodecyl sulfate. Because we needed to accurately compare the concentration of several proteins in brain extracts to investigate the etiology of the disease, the quantitative aspect of immunoblotting was assessed and the results compared for a soluble component with those obtained by electroimmunoassay. Glial fibrillary acidic protein (GFAP) and Tau proteins were analysed by immunoblotting in brain homogenates treated with the Laemmli sample buffer from 10 control and 25 Alzheimer's disease brains. The linearity of densitometric measures of dilutions for one given sample was demonstrated. A 8 to 16-fold GFAP increase in Alzheimer brain was established. With regard to Tau proteins it was possible to show the presence of two pathological Tau variants (Tau 64 and 69) in all the Alzheimer brain homogenates, furthermore, the amount of Tau 64 and 69 was proportional to the presence of neurofibrillary degeneration. As far as alpha 1-antichymotrypsin is concerned, we showed, in a second set of brain samples (14 control and 12 Alzheimer brains), discrepancies between the results obtained by immunoblotting and by electroimmunoassay while for a given sample linearity of immunoblotting measures of dilutions of this sample was demonstrated. Quantitation by immunoblotting of such components which can be quantified using other procedures is uncertain whereas the interest of immunoblotting is undoubted for the insoluble proteins in the brain extracts.

Alzheimer Disease↗
PubMed1988

Alpha 1-antichymotrypsin microheterogeneity in crossed immunoaffinoelectrophoresis with free concanavalin A: a useful diagnostic tool in inflammatory syndrome.

Using crossed immunoaffinoelectrophoresis with free concanavalin A (Con A) in the first dimension and alpha-methyl-D-glucoside incorporated in the second-dimension gel, we examined the microheterogeneity of alpha 1-antichymotrypsin (alpha 1Achy) in sera from healthy donors (N) and in sera from patients with various inflammatory syndromes. We studied three groups: patients with acute inflammation (myocardial infarction: MI, septic inflammation: SI), patients with chronic inflammation (metastatic breast cancer: MBC), and patients with chronic inflammation accompanied by acute attacks (connective-tissue disease: CTD). All pathological sera had a high alpha 1Achy concentration. Compared with N, MI and SI showed an increased proportion of Con A-reactive fraction and a decreased proportion of Con A-nonreactive fraction, which was more pronounced in SI. Unlike the patients with acute inflammation, patients with CTD showed an increased proportion of Con A-nonreactive fraction. Thus alpha 1Achy microheterogeneity in crossed immunoaffinoelectrophoresis may afford a means of differentiating between various inflammatory syndromes. In particular, it can provide a simple test: if the Con A-nonreactive fraction is in a proportion less than 17%, septic origin of an acute-phase reaction may be suspected.

Adult↗
PubMed1987

Studies on the interactions of human pancreatic elastase 2 with human alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin.

The interactions of human pancreatic elastase 2 with alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin were compared by studies in vitro. The equimolar complexes obtained between the enzyme and either inhibitor were relatively stable at 25 degrees C since they could be visualized for up to 5 days by an electrophoretic method. However, in both cases, a slow dissociation occurred with release of active enzyme. As the kass. rate constants are of the same order of magnitude, with a slightly lower value for alpha 1-proteinase inhibitor when compared with alpha 1-antichymotrypsin [(5.6 +/- 1.2) X 10(5) and (8.9 +/- 1.3) X 10(5) M-1.s-1 respectively], partition of human pancreatic elastase 2 between both inhibitors in human plasma is mainly dependent on their respective concentrations. A comparative study by crossed immunoelectrophoresis of the interactions of this enzyme with the two inhibitors contained in normal human plasma and in a mimetic mixture of pure inhibitors was carried out. This allowed the visualization of complexes with either inhibitor. Formation of such a complex with alpha 1-antichymotrypsin had never been demonstrated previously. The patterns obtained are similar when working with normal plasma or with the synthetic mixture, suggesting that, in the conditions used, alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin are the main inhibitors of human pancreatic elastase 2 in the plasma sample. However, it is also shown that part of the enzyme may be taken up by alpha 2-macroglobulin, which is responsible for the remaining enzyme activity on a synthetic substrate. The present work suggests that, according to the delay times of inhibition of human pancreatic elastase 2 calculated from the normal plasma concentrations of alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin, a significant role can be assigned to both inhibitors. Moreover, the role of alpha 1-antichymotrypsin would be enhanced in alpha 1-proteinase-inhibitor deficiency.

Blood Proteins↗
PubMed1987

Identification of proteins by crossed immunoelectrophoresis with a trap-gel.

Simple improvements of the crossed immunoelectrophoresis method are described. A trap-gel was prepared with a small quantity of monospecific antiserum and was submitted to preelectrophoresis. It blocked, during the first dimension, an individual protein as a rocket. The corresponding peak was reduced or disappeared in the second dimension. Identification of precipitation peaks in human or Macacus cynomolgus bronchoalveolar lavage fluids is illustrated and unequivocal recognition of some antigens is shown.

Animals↗
PubMed1987

"In vivo" and "in vitro" inhibition of human pancreatic chymotrypsin A by serum inhibitors.

The interaction of human pancreatic chymotrypsin A with serum inhibitors was assessed by enzyme immunoassay, enzymatic activity and inhibitory capacity measurements and electrophoretic analyses. In normal serum, chymotrypsin A was detected in four forms: one form (Mr approximately equal to 25,000) which might be chymotrypsinogen A and three forms complexed to the main inhibitors present in serum, alpha 2-macroglobulin (alpha 2-M), alpha 1-proteinase inhibitor (alpha 1-PI) and alpha 1-antichymotrypsin (alpha 1-Achy). As chymotrypsin A remains to 90% active when bound to alpha 2-M, the chymotrypsin A/alpha 2-M complex was quantified by an enzymatic assay. The kinetic parameters of the interaction of chymotrypsin A with alpha 1-PI and alpha 1-Achy were determined. Using these data the partition of chymotrypsin A between the different inhibitors in serum was calculated. In acute pancreatitis, the chymotrypsin A plasma level follows the progression of the disease and in this case as well as in normal serum alpha 1-PI is the major antagonist of chymotrypsin A.

Blood Proteins↗