Plasminogen (PLG) polymorphism in northern Japanese: confirmation of PLG*M6 allele

Plasminogen (PLG) phenotyping has been performed on 450 unrelated individuals from northern Japan, using wide-scale ultrathin layer polyacrylamide gel isoelectric focusing combined with immunoblotting. One common phenotype and six rare ones were observed. The rare phenotypes included the recently detected allele PLG*M6 in a new combination with PLG*M5 allele. The estimated allele frequencies for PLG*A, PLG*A3, PLG*M2, PLG*M5, PLG*M6, PLG*B, and PLG*B2 were 0.961, 0.009, 0.001, 0.016, 0.001, 0.003, and 0.009, respectively.     

Phenotyping of alpha-2-HS glycoprotein in bloodstains by isoelectric focusing and immunoblotting

A sensitive immunoblotting procedure has been applied to the detection of alpha-2-HS-glycoprotein (A2HS) phenotypes from control and casework bloodstains. A2HS phenotypes were separated by thin layer polyacrylamide gel isoelectric focusing (PAGIEF) in gels containing Pharmalyte pH 4.2-4.9. After transfer to nitrocellulose by a rapid capillary blot, the A2HS phenotypes were developed using a double antibody enzyme-immunoassay. The evaluation of A2HS phenotyping of casework material was undertaken in parallel with phosphoglucomutase (PGM) phenotyping by PAGIEF. A total of 598 water extracts from casework bloodstains have been tested. Positive results were obtained in 84% and 75% of samples for PGM and A2HS respectively. The A2HS gene frequencies A2HS*1 = 0.6420, A2HS*2 = 0.3530, and A2HS*3 = 0.0050 were determined from a survey of 1000 people in Brisbane.     

中国人血清脱氧核糖核酸酶I(DNase I)的遗传多态性研究

应用薄层PAGIF（T=5％，C=3％）结合特异酶底物染色技术，调查了中国随机人群DNaseI遗传多态性的分布，检出在中国人群中两种常见的等位基因，即DNaseI*1和DNasel*2，其基因频率DNaseI*1为0．53，DNaseI*2为0．47。家系分析表明：子代个体谱带分别来自父亲和母亲，谱带在亲代和子代之间的传递符合孟德尔遗传规律。按Hardy－Weinberg法则进行吻合度检验，观察值与期望值一致。人血清DNaseI等电点经测定为4．0。     

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.     

DIA3 phenotyping in human semen and seminal stains by isoelectric focusing

The polymorphism of DIA3 was investigated by isoelectric focusing in semen samples from 235 unrelated Japanese volunteers and patients. Besides the three common phenotypes seven samples of the type 3-1 were observed. However, readable isoenzyme patterns were not demonstrated in semen samples of oligospermia under about 10 X 10(6)/ml sperm cells. The allele frequencies were DIA3*1 = 0.821, DIA3*2 = 0.164, and DIA3*3 = 0.015. The DIA3*1 frequency in oligospermia (0.765) was lower than that in normospermia (0.836). The isoelectric focusing method was successfully applied to phenotyping DIA3 in seminal stains; each phenotype was demonstrated at 37 degrees C for up to 4 weeks, at room temperature for up to 8 weeks, and at 4 degrees C for over 12 weeks after stain formation. In vaginal swabs the isoenzyme bands were very faint and not identifiable.     

Routine phenotyping of native and desialyzed alpha 2HS-glycoprotein from blood stains

The application of a polyacrylamide gel isoelectric focusing (PAGIEF) and immunoblotting procedure for the identification of native alpha 2HS-glycoprotein (AHSG) in routine casework blood stains has produced reportable results on 57.2% of samples. This reporting rate is lower than that for group specific component (GC) (83.8%) and phosphoglucomutase (PGM 1) (72.8%) phenotyping of the same samples. Blood stain samples were desialyzed with 1 U/ml neuraminidase, overnight at room temperature prior to PAGIEF in gels containing pharmalyte pH 5-6 and 2.5 M urea. Simple AHSG patterns were developed by immunoblotting. This procedure was five times as sensitive as the native AHSG method and desialyzation was reproducible over a range of incubation times and neuraminidase concentrations. The application of the desialyzed AHSG analysis to routine casework samples has resulted in a significant increase in the number of reportable results (762 reported out of 1027 samples). This reporting rate (74.2%) compares favourably with that for GC (79.1%) and PGH 1 (69.6%) phenotyping of the same samples. The three AHSG alleles (AHSG*1, 2 and 3) are clearly resolved after sample desialyzation and separation in gels containing pharmalyte pH 5-6 and 2.5 M urea. The sensitivity of desialyzed AHSG phenotyping approaches that of GC and this technique is worthy of inclusion in blood stain screening protocols of forensic laboratories in regions where the population has a limited range of rare AHSG alleles.     

Molecular analysis of the human esterase D gene ESD(*)Q0(yonago) responsible for incompatibility in a Japanese paternity case

In a Japanese paternity test, an alleged father was excluded only by reverse homozygosity of esterase D (ESD) phenotypes (mother, ESD 1; child, ESD 1; alleged father, ESD 2) out of 43 classical and DNA markers investigated. To solve the aberrant inheritance of the ESD phenotypes observed between them, fragments for all eight coding exons amplified by polymerase chain reaction (PCR) were subjected to DNA analysis. The child and alleged father shared a null allele, originating from ESD(*)1. It was characterized by having TGA for the stop codon instead of TCA for serine at codon 63. Thus, the sharing of a rare null gene, ESD(*)Q0(yonago), increased the probability of paternity.     

The death penalty for juveniles: Bridging the gap between an evolving standard of decency and legislative policy

In Stanford v. Kentucky (1989), the U.S. Supreme Court held that the practice of executing juveniles who were age 16 or 17 at the time of their crime(s) did not violate the “evolving standards of decency” (ESD) of American society. This ESD determination was based on legislative authorization of this punishment. Although this interpretation of what constitutes an ESD has been controlling in death penalty cases since Gregg v. Georgia (1976), the high court's original conception of an ESD stressed the importance of other factors in its determination (e.g., historical review and empirical knowledge about executions). Because the ESD is a Court-created measure, legislatures are under no constitutional obligation to acknowledge the scope of concerns embodied in the historical genesis of this concept. Nevertheless, in this paper we oppose a juvenile death penalty and argue that legislatures should consider the importance of historical and research utilization components of the ESD concept when debating the validity of a policy regarding the death penalty for juveniles.     

Polymorphism of isoenzymes in preserved muscle tissues

In muscles preserved in formalin enzymes were not found to be active. In muscles treated by ethanol the ESD, GLO, GPT and PGP enzymes were active. The best results were obtained in the case of acetone treatment. The phenotypes ESD, GLO, GPT and PGP in tissues corresponded with the ones in the comparative blood samples.     

Determination of phosphoglucomutase (PGM1), acid phosphatase (ACP), and esterase D (ESD) in human bloodstains by hybrid isoelectric focusing (HIEF)

PGM1, ESD, and ACP were determined in bloodstain extracts by isoelectric focusing (IEF) with carrier ampholytes (CA) and HIEF. HIEF yields superior results in PGM typing from bloodstain extracts, whereas for ESD and ACP typing isoelectric focusing with carrier ampholytes seems to be the method of choice.     

用等电聚焦法同步检测血浆类粘蛋白ORM_1亚型和Gc亚型

作者应用等电聚焦加免疫印迹技术，样品经过神经氨酸酶的预处理，首次建立了同步检测血浆ORM1亚型和Gc亚型的分型方法。本法累计个人识别机率为0．8063，累计非父排除率为0．4837。是同步电泳分型中鉴别机率较高的一种。     

HLA 分型用于亲子鉴定10例报告

本文报导了10起亲子鉴定案例的 HLA 分型结果,并进行了遗传学分析和讨论。结果提示 HLA 分型是一种比较准确地解决亲子纠纷的方法。     

The use of ultrathin-layer agarose gels for phenotyping erythrocyte acid phosphatase by isoelectric focusing

A method is described for the use of ultrathin-layer agarose gels in phenotyping erythrocyte acid phosphatase (EAP) by isoelectric focusing (IEF). The results obtained using ultrathin-layer agarose gels are shown to be equally reliable and reproducible in comparison to established ultrathin-layer polyacrylamide gels. IEF of EAP on 0.168-mm agarose gels took place in 90 min using the LKB Multiphor system. The technique described allows for both time and cost efficient phenotyping of EAP.     

红细胞酸性磷酸酶(EAP)型的分布及血痕EAP的检出

本文应用琼脂糖凝胶电泳法对辽宁地区213例汉族随机献血员的红细胞酸性磷酸酶(EAP)型进行了检测,其基因频率为EAP~(?)=0.169,EAP~b-0.831。结合文献资料分析了EAP型分布的种族差异,指出了各人种EAP型分布的特点。用琼脂糖凝胶电泳法,对红细胞溶血液EAP型的最小检出量为2μL(2×3mm滤纸条)及0.5μL(直接加样);对血痕EAP型的最小检出量为7.5μL血液制成的检样。本法在4℃保存4周以内的红细胞溶血液和室温保存25天的血痕均能正确判定EAP的型别。     

Studies on the use of isoelectric focusing as a method of phenotyping erythrocyte acid phosphatase

The technique of isoelectric focusing (IEF) in ultra-thin polyacrylamide gels as a method of phenotyping erythrocyte acid phosphatase (EAP) has been applied to a large number of red cell lysates and dried bloodstains. This paper presents the results of this study and discusses some features of the IEF patterns and problems with their interpretation. The IEF patterns of several rare EAP phenotypes are also described. These studies have confirmed that IEF is more sensitive than starch gel electrophoresis as a method of phenotyping EAP in dried bloodstains.     

The application of immunoblotting to the phenotyping of group-specific component

A sensitive immunoblotting method for the routine detection of group-specific component (GC) from fresh serum, and from control and casework bloodstains has been developed. GC phenotypes were separated in a thin layer polyacrylamide gel by isoelectric focusing, transferred to nitrocellulose by a rapid capillary blotting procedure, and detected using a double antibody enzyme immunoassay. This method is capable of phenotyping 8 ng of GC extracted from bloodstains, a four-fold increase in sensitivity when compared to immunofixation and silverstaining. A total of 2424 casework bloodstains have been analysed and GC phenotypes identified in 78% of samples. The method is suitable for use in routine laboratories and is more sensitive than other methods for GC phenotyping of casework bloodstains.     

Bloodstain characterization in the EAP, Hp, Hb, AK and Glo I typing systems using minigels and the PhastSystem

Application of minigels and the PhastSystem to obtain phenotyping results from bloodstains in the EAP, Hp, AK, and Glo I typing systems was investigated. Nonequilibrium isoelectric focusing with 4-6.5 PhastGel produced readily interpretable phenotypes in the EAP typing system. Both 4-6.5 and 5-8 PhastGel produced AK typing system phenotypes using nonequilibrium isoelectric focusing conditions. The 8-25% PAG PhastGel developed by two staining techniques allowed discrimination of phenotypes for the Hp typing system. Phenotypes from the Glo I typing system were also obtained with this gel type. Variant haemoglobins could be detected on pH 5-8 PhastGel using isoelectric focusing conditions. Much potential for standardized, rapid phenotyping of bloodstains was found to exist utilizing the PhastSystem.     

An investigation into pellet dispersion ballistics.

Existing works on pellet dispersion ballistics are confined to some data-based models derived from statistical analysis of observed patterns on targets but the underlying process causing the dispersion lacks due attention. The present article delves into the relatively unexplored areas of dispersion phenomena, and attempts to develop a theoretical model for general application. The radial velocity distribution of pellets has been worked out by probing into the physical process of dispersion based on transfer of momentum from undispersed shot mass to dispersed pellets. The ratio 2u/v0 (u = root mean square (r.m.s.) radial velocity and v0 = muzzle velocity of the pellets) is found to be fairly constant for a fixed gun-ammunition combination and has been suitably designated as 'Dispersion Index' (DI) characterising its dispersion capability. The present model adequately accounts for pellet distribution on targets and it appears that 'Effective Shot Dispersion' (ESD) as introduced by Mattoo and Nabar [ESD = [(4/N0)sigma Ri2]1/2, where N(0) is the total number of pellets and Ri is the radial distance of the i-th pellet from centre of pattern], gives a faithful numerical measure of overall dispersion at a given distance. A relationship between ESD and firing distance, incorporating the effects of air resistance and gravity has been worked out, which reveals that DI controls the dispersion at a given distance. For small distances (less than 20 m) the relation reduces to a linear one, as already observed empirically and looks like ESD = E0+DI x firing distance, E0 being a parameter dependent on gun and ammunition. The present model, unlike earlier ones, is versatile enough to explain the natures of the dependence of dispersion on firing distance as well as on gun-ammunition parameters, which are essential for a faithful reconstruction of a crime scene. The model has been tested with such experimental data as are available and reasonable agreement is observed.     

Haptoglobin phenotyping of bloodstains by horizontal electrophoresis on a compact polyacrylamide gradient gel

A method is described for phenotyping haptoglobin by horizontal electrophoresis on a small polyacrylamide gradient gel. This method employs the same apparatus used in the separation of many red cell enzyme phenotypes and thereby eliminates the necessity for specialized vertical electrophoresis equipment.     

Analysis of genetic polymorphisms associated with the presence of freckles for phenotypic prediction

The prediction of externally visible characteristics (EVCs) is a commonly used practice by the forensic sciences as an important resource in the investigation of criminal cases in which the identity of perpetrators or victims is unknown or even to recognize decomposed cadavers. With this purpose, genetic markers associated with pigmentation traits have been widely studied by forensic scientists and, nowadays, it is possible to predict phenotypic characteristics such as hair, eyes and skin colour, as well as the presence of skin freckles by analysing single nucleotide polymorphisms (SNPs). In this study, we analysed the association of six SNPs located in pigmentation genes to the presence of freckles in individuals from the Brazilian population for forensic DNA phenotyping. The study was based within the context of a larger project on a population sample of 534 adult Brazilians of both sexes and different skin colours. DNA was extracted from peripheral blood and genotyped using the TaqMan® OpenArray® Real-Time PCR System (ThermoFischer Scientific) technique. Statistical analyses were carried out with the R software (version 4.0.2). As for the results obtained, three SNPs were shown to be statistically associated to the freckling, rs12203592, rs1800404 and rs222847, with CT, AG and AA genotypes being the main contributors, respectively. Variables such as sex of the individuals and skin colour were found to also contribute to the manifestation of this pigmentation trait. Further statistical analyses will be carried out to evaluate the possibility of using the SNPs in this study for phenotyping prediction of the Brazilian population, improving existing DNA phenotyping models in forensic sciences.