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Biomedical subjects

I Mononen

Publications and source records attributed to I Mononen.

At least 55 records · Page 3Linked to original sources
PubMed1992

Prevalence and geographical distribution of major LDL receptor gene rearrangements in Finland.

In order to determine the prevalence of major rearrangements of the low density lipoprotein (LDL) receptor gene in Finland, DNA samples of 199 unrelated Finnish patients with the heterozygous form of familial hypercholesterolaemia (FH) were examined by Southern blot analysis. The FH-Helsinki mutation, characterized by a 9.5-kb deletion in the 3'-end of the LDL receptor gene, was found in 75 (38%) of the patients. The prevalence of this mutation ranged from 26-58% in different areas of Finland. A striking exception was the North Karelia region, where only one out of 26 (4%) FH patients was found to carry the FH-Helsinki allele. Two patients were found to carry other types of large nucleotide rearrangements of the LDL receptor gene. One mutation was a 7.5-kb deletion eliminating exons 7 to 10, and the other was a 13-kb deletion covering exons 11 to 16 of the LDL receptor gene. Serum lipoprotein levels were very similar in each category of mutation, i.e. in patients with the FH-Helsinki gene, those with the two other types of deletion, and the remaining patients with as yet unknown types of LDL receptor gene defects. These results show that, even in genetically uniform populations, FH may be heterogeneous at the DNA level. DNA techniques enable an unequivocal diagnosis for almost 40% of the Finnish patients with the heterozygous form of FH.

Blotting, Southern↗
PubMed1991

Aspartylglycosaminuria in the Finnish population: identification of two point mutations in the heavy chain of glycoasparaginase.

Aspartylglycosaminuria is an inherited lysosomal storage disease caused by deficiency of glycoasparaginase (EC 3.5.1.26) and occurs with higher frequency among Finns than other populations. We have purified human glycoasparaginase and determined about 90% of the amino acid sequence of its light subunit and greater than 70% of that of its heavy subunit by Edman degradation and mass spectrometry. Additional sequence data were obtained from the cloning and subsequent nucleotide analysis of a cDNA corresponding to the normal human glycoasparaginase gene. The enzyme is encoded by a single mRNA as a single polypeptide that is posttranslationally processed to generate the subunits and is glycosylated. After preparing first-strand cDNA from leukocyte and fibroblast total RNA, we used the polymerase chain reaction to amplify the glycoasparaginase cDNA of eight Finnish aspartylglycosaminuria patients. We demonstrate that the Finnish patients' mRNA sequence differed from the normal sequence by two single-base changes six nucleotides apart from one another in the heavy chain of glycoasparaginase. The first change resulted in the replacement of arginine by glutamine (R161Q), whereas the second change resulted in a cysteine to serine substitution (C163S). Both mutations resulted in novel restriction endonuclease sites and were present in all eight Finnish aspartylglycosaminuria patients originating from different pedigrees, but they were absent from Finnish and non-Finnish controls and a non-Finnish case of aspartylglycosaminuria. These results indicate molecular homogeneity in aspartylglycosaminuria alleles in the Finnish population.

Amino Acid Sequence↗
PubMed1991

Glycosaparaginase from human leukocytes. Inactivation and covalent modification with diazo-oxonorvaline.

The apparent active site of human leukocyte glycoasparaginase (N4-(beta-acetylglucosaminyl)-L-asparaginase EC 3.5.1.26) has been studied by labeling with an asparagine analogue, 5-diazo-4-oxo-L-norvaline. Glycoasparaginase was purified 4,600-fold from human leukocytes with an overall recovery of 12%. The purified enzyme has a Km of 110 microM, a Vmax of 34 mumol x l-1 x min-1, and a specific activity of 2.2 units/mg protein with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. The carbohydrate content of the enzyme is 15%, and it exhibits a broad pH maximum between 7 and 9. The 88-kDa native enzyme is composed of 19-kDa light (L) chains and 25-kDa heavy (H) chains and it has a heterotetrameric structure of L2H2-type. The glycoasparaginase activity decreases rapidly and irreversibly in the presence of 5-diazo-4-oxo-L-norvaline. At any one concentration of the compound, the inactivation of the enzyme is pseudo-first-order with time. The inhibitory constant, K1, is 80 microM and the second-order rate constant 1.25 x 10(3) M-1 min-1 at pH 7.5. The enzyme activity is competitively protected against this inactivation by its natural substrate, aspartylglucosamine, indicating that this inhibitor binds to the active site or very close to it. The covalent incorporation of [5-14C]diazo-4-oxo-L-norvaline paralleled the loss of the enzymatic activity and one inhibitor binding site was localized to each L-subunit of the heterotetrameric enzyme. Four peptides with the radioactive label were generated, purified by high performance liquid chromatography, and sequenced by Edman degradation. The sequences were overlapping and all contained the amino-terminal tripeptide of the L-chain. By mass spectrometry, the reacting group of 5-diazo-4-oxo-L-norvaline was characterized as 4-oxo-L-norvaline that was bound through an alpha-ketone ether linkage to the hydroxyl group of the amino-terminal amino acid threonine.

Amino Acid Sequence↗
PubMed1991

High prevalence of aspartylglycosaminuria among school-age children in eastern Finland.

A high prevalence of the lysosomal storage disease aspartylglycosaminuria was found in a study of four birth cohorts of 12882 children in eastern Finland. Using school achievement tests and registers of mentally retarded individuals, 178 mentally retarded children were identified. Randomized urine samples from 151 of the 178 retarded children and from 101 healthy children were analyzed quantitatively for aspartylglucosamine by high-performance liquid chromatography. The results identified three affected individuals in the retarded group indicating an exceptionally high prevalence of aspartylglycosaminuria (1:3643) in the study population, consistent with a carrier frequency of 1:30. The 95% confidence limits for the prevalence are 1:4 352-1:16389. This is the highest prevalence described for any glycoproteinosis in any population and comparable to the incidence figures of the most common lysosomal storage diseases, Gaucher disease type I and Tay-Sachs disease among Ashkenazi Jews. In the study group, aspartylglycosaminuria was, after trisomy 21 (n = 19) and the fragile X syndrome (n = 6), the most common genetic cause for mental retardation.

Acetylglucosamine↗
PubMed1990

Assay of aspartylglycosylaminase by high-performance liquid chromatography.

An aspartylglycosylaminase assay based on high-performance liquid chromatographic analysis of the substrate aspartylglucosamine and product aspartate is described. Aspartylglucosamine and aspartate are derivatized with phenylisothiocyanate and resolved by reverse-phase chromatography. The detection limit for the compounds is 2 pmol. The method can be used for analysis of aspartylglycosylaminase activity in crude cell extracts and tissue samples.

Acetylglucosamine↗
PubMed1989

Analysis of aspartylglucosamine at the picomole level by high-performance liquid chromatography.

A sensitive method for quantitative analysis of aspartylglucosamine as its dabsyl chloride derivative by high-performance liquid chromatography is described. Precolumn-derivatized aspartylglucosamine and internal standard (carboxymethylcysteine) are separated on a reversed-phase column with a mobile phase consisting of phosphate buffer and acetonitrile and monitored by UV-VIS detection at 436 nm. Aspartylglucosamine acts in the assay like a polar amino acid, and it can be separated from interfering substances in urine with a retention time of ca. 13 min. Its detection limit is ca. 0.3 microM in water and 0.5-1.0 microM in urine and other biological samples, which permits its quantitation in normal urine, for example. The within-day coefficient of variation is less than 4.7% and the day-to-day coefficient of variation is less than 8.3%. The present method is applicable to the direct analysis of aspartylglucosamine in body fluids and tissues without any prepurification and, in combination with automated liquid chromatography, allows rapid assay of a large number of samples in the detection of aspartylglycosaminuria. The sensitivity of the assay also allows direct quantitation of aspartylglucosamine in normal urine and leukocytes of aspartylglycosaminuria patients, and may thus be used in metabolic studies of the compound.

Acetylglucosamine↗
PubMed1988

Hemoglobin binding to deglycosylated haptoglobin.

The carbohydrate portion of polymeric haptoglobin was gradually removed by exoglycosidases in order to investigate its role in complex formation between haptoglobin and hemoglobin. Total removal of sialic acid diminished the haptoglobin-hemoglobin complex formation 15%. Removal of about 25% of the galactose residues from asialohaptoglobin, i.e., about 40% of the total weight of the carbohydrate moiety, totally inhibited the ability of haptoglobin to form complex with hemoglobin and react with haptoglobin-specific antibodies. Liberation of further galactose residues resulted in slow precipitation of the protein. Removal of a similar part of the carbohydrate moiety from haptoglobin-hemoglobin complex did not liberate hemoglobin from it, and the complex reacted with haptoglobin antibodies. The combined data indicate that the carbohydrate portion is essential for the functionally active form of polymeric haptoglobin to complex with hemoglobin, but it hardly has any direct role in the binding event, and other factors are responsible for the stability of the complex.

Acetylglucosaminidase↗
PubMed1988

Amniotic fluid glycoasparagines in fetal aspartylglycosaminuria.

Midterm amniotic fluid samples from one pregnancy with the fetus affected by aspartylglycosaminuria and from 11 normal pregnancies were analysed for glycoasparagines accumulating in urine in aspartylglycosaminuria. The aspartylglucosamine concentration in the affected pregnancy was about five times higher than in the controls, but the absolute value was very low being only about one-thousandth of that in urine in aspartylglycosaminuria and one-tenth of that in urine samples from normal adults. In total monosaccharide analysis, only galactose content in the affected amniotic fluid was slightly elevated compared to controls, indicating that higher glycoasparagines typical of urine in aspartylglycosaminuria were not accumulated in significant amounts. The data demonstrate that the analysis of glycoasparagines in amniotic fluid is not likely to permit reliable prenatal diagnosis of aspartylglycosaminuria.

Acetylglucosamine↗
PubMed1988

Laboratory detection of aspartylglycosaminuria.

New specific methods for laboratory detection of a lysosomal storage disease aspartylglycosaminuria were developed. Aspartylglucosamine, the main metabolite accumulating in the body fluids and tissues in the disease, was measured with high-performance liquid chromatography in urine of aspartylglycosaminuria patients, carriers of the disease and normal controls as well as in amniotic fluid of normal pregnancies and one with the fetus affected by aspartylglycosaminuria. In the diseased patients, the aspartylglucosamine excretion was over 1,000-fold elevated compared to that in the carriers and controls. In the pregnancy with the fetus affected by the disease, the concentration of aspartylglucosamine was only slightly elevated, but lower than that in urine of normal individuals. As a conclusion, the determination of aspartylglucosamine in urine allowed postnatal detection of aspartylglycosaminuria, but in amniotic fluid it was useless in prenatal detection of the disease. The activity of the deficient enzyme in aspartylglycosaminuria, aspartylglycosylaminase, was assayed with a specific gas-chromatographic method. The enzyme activity was shown to lack in plasma, lymphocytes and amniotic fluid of aspartylglycosaminuria patients and the method proved applicable for postnatal and prenatal detection of aspartylglycosaminuria. The enzyme activity in lymphocytes of the carriers fell between those in normal controls and aspartylglycosaminuria patients, and the assay could be used in carrier detection in most of the cases.

Acetylglucosamine↗
PubMed1987

Gas chromatography analysis of cellular fatty acids and neutral monosaccharides in the identification of lactobacilli.

Cellular fatty acids and monosaccharides in a group of 14 lactobacilli were analyzed by gas chromatography and the identity of the components was confirmed by gas chromatography-mass spectrometry. From the same bacterial sample, both monosaccharides and fatty acids were liberated by methanolysis, and in certain experiments, fatty acids alone were released by basic hydrolysis. The results indicate that basic hydrolysis gave more comprehensive information about the fatty acids, but the analysis of monosaccharides was found to be much more useful in distinguishing between different species of lactobacilli. The method described allowed differentiation of 11 of 14 Lactobacillus species, and even single colonies isolated from agar plates could be used for analysis without subculturing.

Chromatography, Gas↗
PubMed1986

Detection of sialuria by cation-exchange high-performance liquid chromatography.

A simple and selective method for the detection of sialuria by high-performance liquid chromatography is described. The urine sample (2 ml) is purified using a C18 cartridge or ion-exchange chromatography, and free N-acetylneuraminic acid is separated on an Aminex HPX-87 cation-exchange column using 3 mM sulphuric acid as the mobile phase. The retention time of N-acetylneuraminic acid is ca. 8 min and the detection limit ca. 1 mumol/l. The within-day coefficient of variation is less than 4.9% and the day-to-day coefficient of variation is less than 5.6%. The method was tested on twenty normal individuals and four sialuria patients.

Adolescent↗
PubMed1986

Plasma vitamin C levels are low in premature infants fed human milk.

To evaluate the vitamin C nutritional status of premature infants, plasma vitamin C concentrations were measured in 7 neonates born before 32 wk of gestation and in 13 premature infants born at or after 32 wk of gestation. Samples of umbilical venous plasma from 14 full-term infants were analyzed to provide reference values. Oral feedings with pooled, pasteurized milk from human donors were initiated 1-3 days after birth and intake was gradually increased to 200 ml X kg X day during the second week. After 2 wk, the 13 larger infants received approximately half of their daily milk intake from their own mothers. Results showed that plasma concentrations of vitamin C decline rapidly after birth and approach very low levels in preterm infants fed pasteurized, pooled human milk.

Ascorbic Acid↗
PubMed1986

Determination of cyclic 3'-5'-adenosine monophosphate in plasma by RIA methods in the presence of EDTA.

Calcium ions definitively increase the ability of cyclic 3',5'-adenosine monophosphate (cAMP) to bind to its antibody. In contrast, ethylenedinitrolotetra-acetic acid as its disodium salt (EDTA) shows a dose-dependent inhibition of the binding of cAMP to its antibody. The less sensitive protein binding methods are not affected by EDTA. This is inconvenient, because the EDTA-plasmas can be stored frozen without breakdown of cAMP, but are unsuitable for sensitive radioimmunoassays. The aim of this investigation was to determine how calcium ions and EDTA affect the binding of cAMP to its antibody. Based on these results, we describe an alternative procedure for commercial RIA methods for the determination of cAMP in EDTA-plasma. The almost complete inhibition of the hapten-antibody reaction by EDTA can be abolished by adding an equivalent concentration of calcium ions to the reaction medium together with trichloroacetic acid. Thus a simple and rapid procedure was found for the storage of plasma and for the determination of plasma cAMP.

Calcium↗
PubMed1986

Liquid-chromatographic detection of aspartylglycosaminuria.

We describe a specific, simple liquid-chromatographic method for detecting the lysosomal storage disease aspartylglycosaminuria. The method is based on identification and quantification of the major storage compound 2-acetamido-1-L-beta-aspartamido-1, 2-dideoxy-beta-D-glucose in the urine of affected individuals. Sample preparation and chromatographic analysis requires 30 min. Within-day variation of the method was less than 4.4%, between-day variation less than 11.7% (n = 10 each).

Acetylglucosamine↗
PubMed1985

Isolation and partial characterization of keratan sulphate from human brain.

A sulphated glycoconjugate was isolated from adult human brain from a glycosaminoglycan fraction which was not precipitated with 1% cetylpyridinium chloride or ethanol below 50% concentration. It appeared heterogeneous on gel filtration, exhibiting a molecular weight range from about 7000 to over 10 000. Its main covalent structure was shown to contain sulphated, repeating disaccharide units of (beta-D-galactose-(1----4)-N-acetyl-D-glucosamine-(1----3)). In addition, it was susceptible to degradation by keratan sulphate endo-beta-galactosidase and thus was assumed to be keratan sulphate.

Adult↗
PubMed1985

Adaptation of the May-Grünwald-Giemsa staining method for automated differential counting of blood leukocytes by a Hematrak analyzer.

The selection of exact pH (6.85/6.90) concentrations of the stains and staining times made it possible to use the May-Grünwald-Giemsa (MGG) staining method for smears studied with a Hematrak model 240 automated differential leucocyte counter. The differences between the smears stained by the method of Wright according to the recommendations of the manufacturer and the present MGG-modification resulted in non-significant differences in the recognition of various types of leucocytes. The differences between the manual method and those obtained by Hematrak for the MGG-stained samples with normal total leucocyte counts were nonsignificant with all types of leucocytes except basophils, which were underestimated by the instrument. The present staining procedure was adequate in studying of neutrophilias. When samples with lymphocytosis or with immature cells were examined, too few lymphocytes and immature cells and too many monocytes were obtained by the instrument, as compared to the manual method.

Automation↗