Rare mtDNA haplogroups and genetic differences in rich and poor Danish Iron-Age villages.

The Roman Iron-Age (0-400 AD) in Southern Scandinavia was a formative period, where the society changed from archaic chiefdoms to a true state formation, and the population composition has likely changed in this period due to immigrants from Middle Scandinavia. We have analyzed mtDNA from 22 individuals from two different types of settlements, Bøgebjerggård and Skovgaarde, in Southern Denmark. Bøgebjerggård (ca. 0 AD) represents the lowest level of free, but poor farmers, whereas Skovgaarde 8 km to the east (ca. 200-270 AD) represents the highest level of the society. Reproducible results were obtained for 18 subjects harboring 17 different haplotypes all compatible (in their character states) with the phylogenetic tree drawn from present day populations of Europe. This indicates that the South Scandinavian Roman Iron-Age population was as diverse as Europeans are today. Several of the haplogroups (R0a, U2, I) observed in Bøgebjerggård are rare in present day Scandinavians. Most significantly, R0a, harbored by a male, is a haplogroup frequent in East Africa and Arabia but virtually absent among modern Northern Europeans. We suggest that this subject was a soldier or a slave, or a descendant of a female slave, from Roman Legions stationed a few hundred kilometers to the south. In contrast, the haplotype distribution in the rich Skovgaarde shows similarity to that observed for modern Scandinavians, and the Bøgebjerggård and Skovgaarde population samples differ significantly (P approximately 0.01). Skovgaarde may represent a new upper-class formed by migrants from Middle Scandinavia bringing with them Scandinavian haplogroups.

Human red cell acid phosphatase: purification and properties of the A, B and C isozymes.

Human red cell acid phosphatase isozymes encoded by three alleles (ACP1*A, ACPI*B and ACP1*C), each of which generates two isozymes, (f) and (s), were purified to homogeneity. The molecular mass of the six isozymes (Af, As, Bf, Bs, Cf and Cs) was estimated to be 17-18 kDa, the mass of the f isozymes probably being slightly higher than that of the s isozymes. It was indicated that the isozymes react with p-nitrophenyl phosphate in the mono anionic state, and that a group with a pKa value of about 6, which may be histidine, is of importance for the catalytic function of the s isozymes. Significant differences between the f and s isozymes were observed with respect to specific activity. Km (p-nitrophenyl phosphate), Ki (p-aminobenzylphosphonic acid), amino acid composition, stability in the presence of urea, thermal stability, retention time in size-exclusion chromatography of the native isozymes and migration in sodium dodecyl sulphate polyacrylamide gel electrophoresis, In contrast, identical or similar properties were observed for the three genetically different f isozymes, and the same was the case for the three s isozymes. It is suggested that the f and s isozymes serve different functions in the cell.

Human red cell acid phosphatase (ACP1): the primary structure of the two pairs of isozymes encoded by the ACP1*A and ACP1*C alleles.

The Af, As, Cf and Cs isozymes encoded by the human red cell acid phosphatase ACP1*A and ACP1*C alleles, respectively, have been sequenced. All four isozymes consist of a single non-glycosylated peptide chain (157 residues), acetylated at the amino-terminal alanine residue. Each f isozyme differs from the corresponding s isozyme over the sequence segment 40-73, while the remaining four-fifth of the molecules are identical. These findings are consistent with results for the Bf and Bs isozymes encoded by the common ACP1*B allele and confirm that the presence of a specific f or s segment is a common property to ACP1 isozymes. This supports our hypothesis that f and s isozymes are generated by alternative splicing of exons in the primary RNA transcript. Cf and Cs are identical in sequence with Bf and Bs, respectively. Thus, the ACP1*B and ACP1*C alleles encode exactly the same pair of isozymes, the only difference at the protein level being the ratio of f and s isozyme. Af and As differ from the Bf and Bs isozymes by a single substitution at residue 105; Arg and Gln, respectively. These observations explain the electrophoretic identity of the B and C isozyme pairs and the higher P(i) of the A isozyme pair.

Activity modulation of the fast and slow isozymes of human cytosolic low-molecular-weight acid phosphatase (ACP1) by purines.

The activity modulation of homogeneous isozymes of the human cytosolic M(r) 18,000 acid phosphatase (ACP1) by purines has been investigated. A pronounced difference in the response of fast and slow isozymes of the same genetic type was observed, while identical properties were found for fast isozymes encoded by different alleles (ACP1 X A, B and C), as well as for the corresponding slow isozymes. The catalytic rate constant (kc) of the fast isozymes was increased 5.1-fold by hypoxanthine and decreased 40% by adenine, while the kc of the slow isozymes was unaffected by hypoxanthine but increased 4.6-fold by adenine. This finding and the genetically-determined differences in the relative quantities of the fast and slow isozymes account for the well-known phenotypic differences in activity modulation. The kinetic results strongly indicate that the effector binds to the free enzyme, as well as to the enzyme-substrate complex. Activating effectors showed a higher affinity for the free enzyme than for the enzyme-substrate complex, while the reverse was true with the inhibitor. The results exclude the possibility that effector and substrate bind to the same site of the enzyme; parasteric binding to adjacent sites is suggested.

Phosphonic and arsonic acids as inhibitors of human red cell acid phosphatase and their use in affinity chromatography.

1. In order to obtain an effective ligand for affinity chromatography of the low molecular weight acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) from human red cells nine phosphonic and two arsonic acid substrate analogues were investigated as potential inhibitors. The two forms of acid phosphatase type B (b1 and b2) were isolated and partially purified using conventional methods and the inhibitory action of the substrate analogs investigated. 2. Four of the phosphonic acids were relatively effective competitive inhibitors. It appears that certain structural and electronic requirements have to be fulfilled by the phosphonic acids in order to exhibit significant affinity for the enzyme. A high affinity appears to require the presence of a bulky, hydrophobic moiety which has to be separated from the phosphorus atom by the distance of one atom. 3. p-Aminobenzylphosphonic acid exerted the highest affinity for acid phosphatase with a pH optimum at 6.5. Ki values of 4 . 10(-4) and 6 . 10(-4) M were found for the b1 and b2 forms, respectively. 4. Coupling of p-aminobenzylphosphonic acid to Agarose yielded an effective and specific affinity medium. By means of affinity chromatography using this medium, acid phosphatase was purified 500-fold in a single step.

Hydatidiform mole: genetic markers in diploid abortuses with macroscopic villous enlargement.

In a series of diploid abortion specimens with gross villous enlargement the parental origin was determined by chromosomal heteromorphisms, HLA typing, and enzyme analysis. Diploid androgenesis was the mechanism in 33 cases; in 28 cases the most likely origin was by duplication of a haploid sperm after fertilization of an anucleated ovum, whereas heteromorphisms in five cases indicated a dispermic origin. In one macroscopically complete molar specimen all marker techniques applied indicated a normal conception. In two homozygous specimens a second cell line with both maternal and paternal contributions indicated a twin gestation, whereas, in four conceptuses twinning was suggested solely by HLA determination or ultrasound scan. The observation of a heterogeneous origin of diploid moles, as indicated by three marker systems studied simultaneously, emphasizes that additional information about the frequency of different types of molar conceptions may be obtained by this approach.

Immunochemical characterization of human red cell acid phosphatase isozymes.

An immunological study was performed on human red cell acid phosphatase (ACP1) isozymes encoded by different alleles, each of which is expressed as an electrophoretically fast (f) isozyme and a slow (s) isozyme. These isozymes reacted as two immunochemically different groups. Allele-specific reactions were not detected between either the f isozymes or the s isozymes. Quantitation of ACP1 isozymes in red cells by crossed immunoelectrophoresis revealed a phenotype-dependent variation in the concentration of isozyme protein. A simple gene dosage effect was indicated and the ordering of the ACP1 alleles (ACP1*A less than ACP1*B less than ACP1*C less than ACP1*E) was identical to that found for enzyme activity levels. Also, an allele effect on the proportion between s and f isozymes (s/f) was observed; the ordering here was ACP1*B less than ACP1*A less than ACP1*C, which is the same as that reported for the susceptibility to modulation with purines. These variations in isozyme protein levels appear to account for the phenotypic differences in the intensity of the isozyme bands, when activity-stained after electrophoresis, and in the red cell enzyme activity levels. Investigation of two carriers of a Null allele showed no evidence of an aberrant protein product, and half-normal concentrations of enzyme protein were observed in the red cells of these individuals.

Human red cell esterase D polymorphism in Denmark, its use in paternity cases and the description of a new phenotype.

Red cell esterase D (EsD) phenotypes were determined in a Danish population sample of 3,116 unrelated adults by starch-gel electrophoresis. A new phenotype was discovered, which appeared to be determined by the EsD1 allele and a new allele EsDCph. The gene frequencies observed were EsD1 = 0.9007, EsD2 = 0.0992, EsDCph = 0.0001. Investigation of 1,111 mother-child pairs and 59 families with 157 offspring added further support to the genetic model of two common alleles at an autosomal locus. The applicability of the EsD polymorphism to paternity testing was investigated on 960 cases of disputed paternity. An estimate of the EsD null allele frequency (0.001) in European populations was made on the basis of observations made on 5,864 mother/child combinations and 762 matings with 1,882 offspring. The influence of this allele on the reliability of exclusions of paternity was determined.

Human red cell acid phosphatase: quantitative evidence of a silent gene PO, and a Danish population study.

In a forensic case of disputed paternity an apparent mother/child incompatibility with respect to red cell acid phosphatase was found, the mother appearing as type A and the child as type B. Determination of electrophoretic type and of acid phosphatase activity in 8 of the family members strongly suggested the presence of a silent gene PO in 4 of the individuals. The phosphatase levels in the four heterozygotes were about half the values expected from normal values determined in 100 healthy adults representing the different phenotypes. The distribution of red cell acid phosphatase types in 3,735 unrelated Danish adults and in 1,109 mother/child pairs is reported; gene frequencies Pa = 0.369, Pb = 0.566 and Pc = 0.065. The PO gene frequency was roughly estimated as 0.001. Results are reported on the application of the red cell acid phosphatase system to 300 2-men cases of disputed paternity.

Association between the C3F gene and atherosclerotic vascular diseases.

The C3 phenotype distribution was studied in a group of patients suffering from atherosclerotic vascular diseases. A statistically significant association was found between the presence of the C3F gene and the occurrence of atherosclerosis. A relative risk incidence of the disease of 1.87 was found for the C3F-positive individuals as compared to the C3F-negative ones.

C3 polymorphism in relation to age.

The C3 phenotype distribution was investigated in different age-groups among 2,078 voluntary blood donors between the ages of 20 and 65 years, in a group of unrelated babies and in a group of old healthy persons. A continuous increase in the C3F gene frequency with age was found among the blood donors varying from 0.1780 in the youngest age group (babies: 0.1585) to 0.2516 at the age of 50-55 years followed by a continuous decrease to a level of 0.1700 among the eldest donors (01718 among the old persons). In the age group 45-49 years the C3 distribution differed significantly from that in the adjoining age-groups (C3F = 0.1619). It is believed that the variations are brought about by selection of the blood donor population and a balanced polymorphism for the C3 system, possibly due to differences in the biological efficiency of the C3 variants in the complement sequence.

Human red cell galactose-1-phosphate uridylyltransferase (EC 2.7.7.12). Electrophoretically determined polymorphism in Denmark and its use in paternity cases.

The electrophoretically detectable phenotypes of human red cell galactose-1-phosphate uridylyltransferase (GALT) were determined in 2,074 unrelated Danes. The gene frequencies were: GALT1 = 0.9233 and GALT2 = 0.0767. The segregation of phenotypes in 765 mother-child pairs was consistent with autosomal codominant inheritance. One apparent mother-child incompatibility with respect to phenotypes was observed which, however, appeared to be due to the segregation of a silent gene. The results of an investigation of 248 paternity cases are reported, and the application of the GALT polymorphism to paternity cases is discussed.

Simultaneous detection of ACP1 and GC genotypes using PCR/SSCP.

The classical enzyme and protein markers ACP1 and GC have gained new importance because of the biological functions of their gene products. ACP1 encodes a low molecular weight enzyme which is now recognized as a phosphotyrosine phosphatase with a role in the regulation of signal transduction pathways, and GC-globulin acts both as a transporter of vitamin D and as a plasma actin scavenger and plays a role in macrophage activation. These two polymorphisms were phenotyped for decades on the basis of electrophoretic isozyme or protein patterns; the gene structures are now known. Nucleotide substitutions determining the common alleles are close enough at each locus to be contained in one short PCR product. We have developed a simple, rapid and reliable multiplex method based on PCR and SSCP which allows the simultaneous determination of the common ACP1 and GC genotypes.

Delirium tremens: some clinico-chemical features. A study of alanine-aminotransferase, alcaline phosphatase, prothrombine and enolase.

The relationship between variables reflecting liver disease (serum-alanine-aminotransferase (SGPT), serum alcaline phosphatase and plasma prothrombine) and the clinical signs and symptoms during delirium tremens (DT; grade 3) and related clinical states (grade 2) was studied. Furthermore, it was investigated whether the two isoenzymes of enolase which predominante in brain tissue were present in plasma or cerebrospinal fluid (CSF) in DT patients. A correlation between SGPT and clinical state was not observed, which indicates that a causal relationship does not exist between acute liver cell damage and clinical state during DT of grade 3 or 2. In grade 2 patients, but not in grade 3 patients, both SGPT and serum alcaline phosphatase decreased between admission and recovery. This difference between the groups may be due to a higher alcohol consumption and a shorter interval between last drink and admission in grade 3. The difference in recent drinking history may also account for the finding of a higher plasma prothrombine index in grade 3 compared with grade 2, because chronic ethanol intoxication may be accompanied by enhanced hepatic protein synthesis. "Brain-enolase" was not present in detectable amounts in blood or CSF during DT thus suggesting that brain cell damage resulting in leakage of this enzyme from the cells did not prevail during DT.

Human red cell acid phosphatase (ACP1): evidence for differences in the primary structure of the two isozymes encoded by the ACP1*B allele.

Molecular properties of the two isozymes expressed by the B allele at the red cell acid phosphatase locus (ACP1) have been studied to distinguish between possible mechanisms for their production. The difference in electric charge exhibited by the native isozymes was retained under denaturing conditions; the unfolded peptide chains renatured without conversion of one form to the other. Chromatographic analysis [thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC)] of tryptic digests showed 12 peptides common to both isozymes but also revealed 5 peptides unique to one isozyme and 3 (possibly 4) peptides unique to the other. These findings argue against both conformational isomerization and simple posttranslational modification as the mechanism of generation of the two isozymes. We suggest that the two isozymes are synthesized as discrete molecular entities.

Human red cell acid phosphatase (ACP1). The amino acid sequence of the two isozymes Bf and Bs encoded by the ACP1*B allele.

The pair of isozymes, Bf and Bs, encoded by the human red cell acid phosphatase ACP1*B allele has been sequenced. Similar but not identical primary structures were observed. Both isozymes consist of a single peptide chain of 157 amino acid residues, which is acetylated at the amino-terminal alanine residue. The Bf and Bs isozymes are not glycosylated, and the calculated molecular masses are 17,932 and 17,867 Da, respectively. They are identical except for the sequence segment 40-73, which is peculiar to the respective isozyme. This is consistent with our hypothesis that the two isozymes are generated as the result of alternative splicing of the primary RNA transcript. The finding of a signature sequence offers the basis for the characteristic differences in catalytic and molecular properties of the Bf and Bs isozymes. A high degree of homology was found between the Bs isozyme and the 18-kDa cytosolic acid phosphatase from bovine liver. No homology was observed with other sequenced proteins, and this establishes these low molecular weight acid phosphatases as products of a distinct gene family.

Human 18 kDa phosphotyrosine protein phosphatase (ACP1) polymorphism: studies of rare variants provide evidence that substitutions within or near alternatively spliced exons affect splicing result.

The mammalian low molecular weight phosphotyrosine protein phosphatase is expressed as two distinct isoforms. The human 'fast' and 'slow' isoforms differ only in the sequence of an internal segment of 34 residues, and the ACP1 gene contains two adjacent exons (E3F and E3S) which encode these segments. We have previously suggested that the fast and slow isoforms are generated by mutually exclusive pre-mRNA splicing of E3F and E3S. The common alleles ACP1*A, *B and *C express the fast and slow isoforms in different ratios. The *A and *C alleles differ from *B by C --> T transitions in E3S and E3F respectively. To test the idea that the fast : slow ratio is determined by nucleotide substitutions in the E3F-I3F-E3S region, four groups of rare ACP1 variants with unusual fast : slow ratios and the rare *E and *R alleles, expressing fast∶slow ratios similar to *C and *B, respectively, were analysed. Gene segments of the I2-I3S region were amplified by PCR and analysed by SSCP and variant bands were excised and sequenced. For each of the rare isozymic variants one of six different nucleotide substitutions in E3F (nts+42, +85, +109, +110), I3F (nt+1) and I3S (nt+8) was observed. The *E and *R alleles showed C and B sequence, respectively, in accordance with the fast : slow ratio. The results support the hypothesis that the fast : slow ratio is constitutive.

Exon structure at the human ACP1 locus supports alternative splicing model for f and s isozyme generation.

The human ACP1 locus encodes a genetically polymorphic cytoplasmic low-molecular-weight acid phosphatase. Each of the common alleles encodes two isoforms, f and s. Both isozymes are of equal length (157 residues) but differ in sequence over an internal 34 residue segment. Substantial portions of the ACP1*A, *B and *C alleles common to Europeans have been sequenced. Six linearly positioned exons containing codons 14 to 157 were identified. Two exons of equal length (114bp) interspaced by a short (41bp), probably nonfunctional, intron encode the specific f and s segments, respectively. These findings strongly support an alternative RNA splicing hypothesis. In addition, three allele-specific base substitutions were encountered.

Rapid and simple determination of hereditary haemochromatosis mutations by multiplex PCR-SSCP: detection of a new polymorphic mutation.

Hereditary haemochromatosis is a common inherited disorder leading to excessive accumulation of iron in various organs. Two missense substitutions at the HFE-gene have recently been associated with the disease, 187C G and 845G-->A (mutations H63D and C282Y, respectively). We present a simple, rapid PCR-SSCP multiplex screening method allowing the simultaneous detection of both substitutions. Furthermore, testing the method on 420 Danish blood donors revealed the presence of a hitherto undetected third substitution in 13 individuals. The new substitution, a 193A-->T transversion, affects codon 65 changing the code for serine to that of cysteine (S65C). It may thus have functional consequences for the HLA class protein encoded by the HFE-gene. The allele frequencies observed were: H63D 14.8%, C282Y 6.2% and S65C 1.5%, which for the two former alleles are in agreement with frequencies reported for other North European population samples.