Plasma protein and red cell enzyme groups in Galicia (north west Spain)

Galactose-1-phosphate uridyl transferase (GALT), esterase D (EsD), and plasminogen (PLG) phenotypes were determined by isoelectric focusing in thin-layer polyacrylamide gels (PAGIF) in a random sample from Galicia. Haptoglobins (Hp) were determined by conventional electrophoresis. The following gene frequencies were observed: for GALT: GALTN: 0.930; GALTD1: 0.044; GALTD2: 0.025; for EsD: EsD1: 0.874; EsD2: 0.104; EsD3: 0.021; for PLG: PLG1: 0.800; PLG2: 0.199; for Hp: Hp1: 0.426; Hp2: 0.573. Population data results of all electrophoretic markers typed until now in Galician population are also included.     

Polymorphism of EsD by isoelectric focusing: description of the new allele EsD*Kofu and phenotyping in bloodstains

The polymorphism of EsD was investigated in 1115 unrelated Japanese individuals by isoelectric focusing. Besides the three common phenotypes two heterozygotes EsD 7-1 and EsD 7-2 were observed. The gene frequencies were: EsD*1 = 0.6234, EsD*2 = 0.3663, and EsD*7 = 0.0103. In addition, a rare variant was detected in a probandus living in the city of Kofu. The family analysis suggested the hereditary occurrence of a new allele EsD*Kofu. The isoelectric focusing method was successfully applied to phenotyping EsD in bloodstains; each phenotype was demonstrated at 37 degrees C for up to 2 weeks, at room temperature for up to 9 weeks, and at 4 degrees C for over 20 weeks after stain formation.     

The use of isoelectric focusing (IEF) as a method for the combined phenotyping of erythrocyte acid phosphatase (EAP) and esterase D (EsD)

The simultaneous isoelectric focusing (IEF) in polyacrylamide gels (PAG) of erythrocyte acid phosphatase (EAP) and esterase D (EsD) allows the poor discriminating power (DP) of EsD to be usefully combined with a highly discriminating system EAP, such that a joint DP of 0.766 was achieved compared with PGM IEF DP 0.756. Focusing was carried out in a centrally flattened gradient containing ampholines (pH 4-6 and 6-8) and the chemical spacer 3-(N-morpholino) propanesulphinic acid (MOPS). It enabled the identification of six EsD phenotypes including the recently discovered EsD5 isozymes. The application of this method to casework bloodstains is discussed.     

Genetic study of red cell esterase D polymorphism by ultrathin layer isoelectric focusing. Distribution in the Veneto population (Italy)

Human red cell Esterase D (EsD) was analyzed by isoelectric focusing (IEF) on ultrathin-layer polyacrylamide gel with a pH range of 5.0-6.0. Hemolysates were treated with Dithiothreitol to avoid loss of activity and change of the isozyme patterns by in vitro storage effects. In our sample of 951 unrelated persons from Veneto, seven different phenotypes were observed. The following allele frequencies were calculated: EsD1 = 0.8476, EsD2 = 0.1336, EsD5 = 0.0178, and EsDV = 0.0010.     

EsD*5 gene frequency in Tuscany (Italy)

The distribution of the human red cell esterase D (EsD) "extended" polymorphism in a population sample from Tuscany (Italy) was studied using agarose gel isoelectric focusing. The estimated gene frequencies were: EsD*1 0.864, EsD*2 0.115, EsD*5 0.021. The EsD*5 allele frequency is very similar to those reported for other European populations. The "extension" of the EsD polymorphism may prove to be useful in paternity testing.     

Polymorphisms of the enzyme systems galactose-1-phosphate uridyltransferase (GALT) and esterase D (EsD) in the province of Cádiz, southern Spain

Galactose-phosphate uridyltransferase (GALT) and esterase D (EsD) phenotypes were determined by isoelectric focusing in ultrathin-layer polyacrylamide gel (PAGIF) for 406 healthy subjects randomly chosen and residing in the province of Cádiz, in Southern Spain. The following gene frequencies were observed: for GALT, GALT1 = 0.952 970 3 and GALT2 = 0.047 029 71; for EsD, EsD1 = 0.895 320 2, EsD2 = 0.094 827 59, and EsD5 = 0.009 852 21.     

Evidence for a "new" allele at the esterase D (E.C. 3.1.1.1.) locus

An apparently new EsD gene product (EsD*Düsseldorf) was detected by use of horizontal agarose gel electrophoresis (AGE), starch gel electrophoresis (SGE), and isoelectric focusing (IEF). The observed phenotype EsD (1-Düsseldorf) can be distinguished from any known EsD type.     

Esterase D typing of bloodstains by non-equilibrium focusing

Non-equilibrium focusing in a pH 4-6 gradient in ultra-thin polyacrylamide gels has been shown to be a reliable and reproducible method for detecting the six common esterase D phenotypes (EsD 1,2-1,2,5-1,5-2 and 5) in dried bloodstains. Successful typing is dependent on both the age and phenotype of the stain in question. The effects of age on the isozyme pattern of each phenotype are described and illustrated. In a comparative trial using 100 simulated and 300 authentic casework bloodstains, non-equilibrium focusing was shown to be more efficient than thin-layer starch gel electrophoresis for the typing of esterase D.     

Polymorphism of EsD by isoelectric focusing: phenotyping in human tissues, dental pulps, hair roots, and semen

The polymorphism of EsD was investigated in tissues of various human organs, dental pulps, hair roots, and seminal stains by isoelectric focusing. The method yielded an excellent resolution of the isoenzyme components. The time limits of determination were: in organ tissues 3 weeks, in dental pulps 1 week, and in hair roots several days. The 7-1 type was less stable than the common types. Phenotyping was possible from fresh semen samples, but was unsuccessful from dried seminal stains after storage. The results show that the EsD typing by isoelectric focusing is of practical use for medicolegal individualization of organs, teeth, and hairs.     

Non-equilibrium pH gradient electrophoresis (NEPHGE) on ultrathin polyacrylamide gels containing separators: improved erythrocyte phosphoglucomutase (PGM) and esterase D (EsD) diagnosis in red cell lysates and bloodstains

Two rapid and reliable electrophoretic techniques for PGM1 and EsD typing on ultrathin polyacrylamide gels are described. They have been based on non-equilibrium pH gradient electrophoresis and on the addition of chemical spacers (EPPS for PGM1 and HEPES for EsD) to the gel mixture.     

红细胞EsD和PGM_1的同步电泳分型及其在上海地区的分布与频率

用 pH7.4的 Tris-马来酸缓冲系统和混合淀粉凝胶同步检测血液及血癌中 EsD 和 PGM_1的表型,获得良好的分型效果。EsD 和 PGM_1的图谱区带平直、狭窄、清晰。各种表型之间差异著,极易区分容易发现稀有表型。我们在上海地区居民中检查了390人的 EsD 表型和724人的 PGM_1表型,其分布与其基因频率详见附表。在检测尸体血及尸体血痕时,发现一例尸体血和一例尸体血痕的 PGM 1活性明显增强,前者尚显现了一条额外的同工酶区带。     

The use of separator isoelectric focusing in micro-ultrathin polyacrylamide gels in the characterization of some polymorphic proteins of forensic science significance

The identification of phenotypes of erythrocyte acid phosphatase (EAP), esterase D (EsD), group specific component (Gc), and alpha-1-antitrypsin (PI) by separator isoelectric focusing in micro-ultrathin polyacrylamide gels (interelectrode distance: 45 mm) is described. The protein patterns obtained are compared favorably with the patterns seen by isoelectric focusing in conventional polyacrylamide gel dimensions (interelectrode distance: 110 to 120 mm). The technique described allows greater stability of pH gradients and is a fast and economic method.     

Evaluation of a nonequilibrium isoelectric focusing (IEF) method for the simultaneous typing of esterase D (EsD), red cell acid phosphatase (AcP1), phosphoglucomutase (PGM1), adenylate kinase (AK), and adenosine deaminase (ADA)

A nonequilibrium isoelectric focusing method incorporating the chemical spacers MOPS and HEPES was developed and subsequently evaluated for its ability to reliably discriminate common and rare phenotypes in the esterase D (EsD), red cell acid phosphatase (AcP1), phosphoglucomutase (PGM1), adenylate kinase (AK), and adenosine deaminase (ADA) isoenzyme systems. The validation procedures used were blind testing, comparison of results to conventional methods, and evaluation of known rare variant phenotypes. This method proved to be a quick and reliable method for typing all five isoenzyme systems, while providing an excellent probability of discrimination (PD = 0.96).     

Paternity testing by using erythrocyte enzyme esterase D.

A total of 206 paternity cases were tested for the erythrocyte enzyme esterase D (EsD) along with the 15 genetic marker systems routinely phenotyped. Of 39 observed exclusions of falsely accused males, 5 were exonerated by EsD. Reliability and ease of testing indicates that EsD is a useful exclusion determinant.     

用地高辛配基标记的pAW101探针杂交分析DNA多态性及其应用

将NaCl盐析法抽提人基因组DNA与地高辛配基标记DNA探针的方法相结合，检测了15个家系子代与亲代DNApAW101－EcoRI限制性片段的遗传关系。结合所测同工酶（EsD．GLOI．pGM；．Acp）的基因频率、按Essen－moller氏公式计算，父权概率均达到99．73％以上。本法经济、有效、实用、易于在国内普遍实验室开展。     

Population genetic examination of esterase D in the Lübeck area (author's transl)]

1251 blood samples of non-related human beings coming from the area of Lübeck have been examined. Frequencies of the esterase D allel were EsD1 = 0.9001 and EsD2 = 0.0999.     

Frequencies of red cell enzyme polymorphisms acP, ADA, AK, EsD, 6-PGD, and PGM1 determined by parallel investigations of Turks and Germans living in the Lübeck area (author's transl)

Gene frequencies for enzyme polymorphisms in the acP, ADA, AK, EsD, 6-PGD, and PGM1 systems were determined by a random sample (n = 281-556-575) Turks living in Lübeck. The results were compared with those of a parallel inquiry on Germans from Lübeck. The following gene frequencies were detected: (table: see text).     

A new 39-plex analysis method for SNPs including 15 blood group loci

A novel 39-plex typing system for single nucleotide polymorphisms (SNPs) has been developed. This multiplex approach has the advantage of being able to type 38 autosomal SNPs and one sex-discriminating base exchange site on the X and Y chromosomes rapidly and simultaneously. The SNP loci on the autosomes, which we examined, contain 15 loci distributed on blood type genes: three on RhCE, two each on Km and Gc, and one each on Duffy, AcP1, Tf, MN, GPT, EsD, PI, and Kidd genes. Thirty-seven genomic DNA fragments containing a total of 38 SNPs and one sex-discriminating site were amplified in one multiplex PCR reaction. Following the reaction, single nucleotide primer extension reaction was performed by dividing these SNP loci into five groups. The SNP type of each of the 39 loci was determined at one time by capillary electrophoresis using the newly designed multi-injection method. The combined PD (power of discrimination) of this typing system was (1-1.1) x 10(-14), and the MEC (mean exclusion chance) was 0.9990. We applied this system to forensic cases, including 16 paternity testing cases (13 non-exclusion and three exclusion cases) and one personal identification case. For the paternity testing cases, the highest Essen-M?ller's W-value was 0.9999995. The pM (matching probability) of the personal identification case was 2.22 x 10(-17). These data showed that this system was an excellent tool for use in forensic cases of paternity testing and personal identification.     

New detection method for ribonuclease 2 (RNase 2) using immunoblotting with specific antibody

A ribonuclease (RNase) was isolated from the urine of a 35-year-old male and purified to electrophoretic homogeneity. The enzyme was tentatively designated RNase 2. A rabbit antibody produced by injection of the purified RNase 2 was able to distinguish RNase 2 from another type of RNase coexisting in body fluids. With this antibody it was possible to detect RNase 2 isozymes in human serum and urine without difficulty using isoelectric focusing or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting. Both RNase 2 in serum and urine seemed to exist in multiple forms with regard to their molecular masses and pI values. This technique may prove to be useful in genetic and forensic studies of RNase polymorphism.     

Isoelectric focusing of human hair keratins: correlation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and effect of cosmetic treatments.

A new isoelectric focusing (IEF) technique in polyacrylamide gels with 6M urea and 1.5% Nonidet P40 has been developed to characterize human hair samples. The phenotypes demonstrated with this procedure has been correlated with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns described by other authors. The method described can be applied in the forensic science analysis of a single human hair. Using the same IEF technique we have studied the changes in electrophoretic patterns of cosmetically treated hair. The characteristics of the modifications observed and its utility in forensic science work are also discussed in this paper.