Simultaneous electrophoretic determination of phosphoglucomutase subtypes, adenosine deaminase, erythrocyte acid phosphatase, and adenylate kinase enzyme phenotypes

Many of the conventional agarose phosphoglucomutase (PGM) subtyping systems presently in use fail to provide a good separation between the 1 + and 2- bands as well as the 2+ band and the more anodic moving bands. Use of a 1-mm-thick gel composed of 1% ISO GEL (FMC Corp.) and phosphate-citric acid gel and tank buffers with a pH of 5.3 provided exceptionally good separation between all four of the major subtyping bands. The additional criteria for this procedure is a voltage of 21 V/cm and a run time of 4 h. Utilization of this procedure using case samples of varied ages proved the reliability of the procedure. Also examined were the effects of several reducing agents on the enzyme band patterns and the use of this system for the simultaneous determinations of the adenosine deaminase (ADA), erythrocyte acid phosphatase (EAP), and adenylate kinase (AK) enzyme phenotypes.     

Phosphoglucomutase types in blood and hair roots taken from post-transfusion subjects

Pre- and post-transfusion blood samples were collected from 22 subjects together with the corresponding plucked hair samples taken 2 days and 2 weeks after the transfusion. The phosphoglucomutase1 (PGM1) subphenotypes of blood and hair were determined by isoelectric focusing and the phenotypes confirmed by gel electrophoresis. Many of the post-transfusion blood samples showed an alteration in the PGM1 bands when compared with the pre-transfusion samples. However, the PGM1 types determined from the hair samples were identical to the corresponding pre-transfusion samples in all cases.     

Simultaneous phenotyping of EAP and PGM1 by PAG1F

The study is to find new genetic markers of simultaneous phenotyping and to raise the determinating power of isoenzymes. Simultaneous electrophoresis of EAP and PGM1 by thin-layer PAG1F combined with reverse zymogram developing was applied and clear isoenzyme bands was observed on both surface of a gel without interference. The method can do typing of the EAP and PGM1 at one gel and the accumulative DP is 0.87. It can be used in paternity test.     

Studies and observations on the use of isoelectric focusing in ultra-thin polyacrylamide gels as a method of typing human red cell phosphoglucomutase

The technique of isoelectric focusing in ultra-thin polyacrylamide gels as a method of typing human red cell phosphoglucomutase (PGM1) has been studied. Typing was possible without the samples attaining true equilibrium focusing conditions. The isozyme patterns so obtained were clearly defined and free from distortion. The importance of assessing relative band intensities when interpreting the isozyme patterns is discussed. Our experience of using the technique to analyse casework material is described.     

Unusual results due to transfused blood.

During a murder enquiry, the forensic science investigation used PGM and EAP blood grouping systems and detected a mixture of blood on the deceased's jacket. The blood groups matched those of the deceased and accused. The results of DNA analysis, however, proved that only a single source of DNA, matching the deceased, was present. Supplementary information relating to the transfusion of the individual whilst still wearing his clothing led the authors to conclude that a mixture of transfused blood and the individual's own blood had effused from his body via a stab wound, and onto his clothing.     

Blood group frequencies of the population of Trinidad and Tobago, West Indies.

The results of grouping tests performed on blood samples collected over a five-year period at the Trinidad and Tobago Forensic Science Centre are presented. The samples were tested using the ABO, PGM, EAP and GLO blood group systems. Phenotypic frequencies and allele frequencies for each system were calculated for the two major ethnic groups of the population, the African and East Indian. Matching probabilities, which can be used in the interpretation of physical evidence in forensic cases, were also calculated.     

A study of independence between STR and conventional blood type loci

Blood samples from approximately 200 Scottish Caucasian individuals were typed at conventional loci (PGM, Gc and EAP) and also with a four locus STR multiplex. Tests of the data are described which demonstrate that the assumptions of between locus independence are robust for use in forensic casework.     

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。     

Genetic markers in semen. III: Alteration of phosphoglucomutase isozyme patterns in semen contaminated with saliva

Contamination of semen by saliva can result in the alteration of seminal phosphoglucomutase (PGM1) isozyme patterns. The alteration is characterized by the gradual loss of the a and b isozyme bands the concomitant generation of anodal bands; eventually, all PGM activity is lost. The conversion of PGM isozyme patterns has been shown to be due to a dialyzable heat-labile factor in saliva and a nondialyzable heat-labile factor in semen. The implications of this conversion for PGM typing in sexual assault evidence are discussed.     

The evaluation of five electrophoretic phenotyping systems for routine screening of bloodstains

The performance of the polymorphic marker systems group-specific component (GC), phosphoglucomutase-1 (PGM), alpha-2-HS-glycoprotein (A2HS), haptoglobin (Hp), and erythrocyte acid phosphatase (EAP) was evaluated on control bloodstains. The major factors considered were: sensitivity of the test system; stability of the marker; laboratory economics of each test; and distinguishing power (Dp) of the system. GC was considered to be the most suitable marker for routine screening because of its high stability and Dp, and the sensitivity of the immunoblotting detection method. PGM and A2HS were the next most valuable markers followed by Hp. EAP could only be considered useful when large amounts of relatively fresh bloodstain were available.     

Ribosomal ribonucleic acid (rRNA) gene typing for species identification.

Deoxyribonucleic acid (DNA) typing of ribosomal ribonucleic acid (rRNA) genes was performed with a polymerase chain reaction (PCR) assay for species identification. A variable region of the 28S ribosomal RNA gene was amplified with primers complementary to flanking sequences phylogenetically well conserved. The products of twelve animal DNAs (human, Japanese monkey, dog, cattle, pig, cat, rabbit, mouse, rat, chicken, frog, and fish) were separated by polyacrylamide gel electrophoresis, each revealing a few bands ranging from 150 to 100 base pairs. The band patterns obtained from each DNA sample differed in number and size, which indicates the applicability of the method to species identification. Samples containing either as little as 1 pg of DNA or degraded DNA of 0.2 to 0.5 kb in length were able to give detectable bands. Postmortem human tissue DNAs were tested as an example. They showed a pattern identical to the human control one, which was distinct from those of the other animals examined.     

用国产两性电解质进行血痕的种属鉴定

本文应用国产两性电解质等电点聚焦法(PAGIEF)对1个月的40种动物血痕浸出液及经PCMB处理后的血痕浸出液的谱型进行了研究。根据谱型特征,成人除与人胎儿及猴难以鉴别外,与其它动物均可鉴别。动物间除鸟纲,鱼纲内有五组动物组内难以鉴别外,其它均能鉴别。经PCMB处理后,成人与人胎儿的谱型仍无差别,与其它动物的鉴别则更容易。结果表明,国产两性电解质用于等电聚焦进行血痕的种属鉴别是完全可能的。     

Sex identification in fresh blood and dried bloodstains by a nonisotopic deoxyribonucleic acid (DNA) analyzing technique

Deoxyribonucleic acid (DNA) specimens were prepared from blood or bloodstain extracts, and the content of a Y-chromosome specific DNA fragment was investigated by the Southern hybridization method using a nonisotopic staining technique. Thus obtained patterns of male DNA showed a clear band, whereas broad stains with some faint bands appeared on the patterns of DNA from both sexes. This method is expected to be a new powerful mean of forensic medical examination.     

The use of isoelectric focusing to identify rhinoceros keratins.

Keratins represent the principal structural proteins of hair. They are also found in horn, nail, claw, hoof, and feather. Hair and nail samples from human and canine sources and hair samples from mule deer, white tail deer, cat, moose, elk, antelope, caribou, raccoon, and goat were studied. Parrot and goose feathers were also analyzed. Keratins are polymorphic, and species differences are known to exist. Proteinaceous extracts of deer and antelope antlers and bovine and rhinoceros horn were prepared by solubilizing 10 mg of horn sample in 200 microL of a solution containing 12M urea, 74mM Trizma base, and 78mM dithiothreitol (DTT). Extraction took place over a 48-h period. A 25-microL aliquot of extract was removed and incubated with 5 microL of 0.1 M DTT for 10 min at 25 degrees C. Keratins were then separated by isoelectric focusing (IEF) on 5.2% polyacrylamide gels for 3 h and visualized using silver staining. At least 20 bands could be observed for each species studied. However, band patterns differed in the position of each band, in the number of bands, and in band coloration resulting from the silver staining process. Horn from two species of rhinoceros was examined. For both specimens, most bands occurred in the pH range of 4 to 5. Although similar patterns for both species were observed, they differed sufficiently to differentiate one from the other. As might be expected, the closer two species are related phylogenetically, the greater the similarity in the IEF pattern produced from their solubilized keratin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polymorphism of seminal gamma-glutamyl transpeptidase

Electrophoretic analysis of seminal gamma-glutamyl transpeptidase (GGT) activity of 147 unrelated Japanese males revealed three types of band patterns. An anodal single band, a cathodal single band and heterozygous double bands termed 1, 2 and 2-1, respectively, were commonly identified in the samples. The frequencies of the three types were 1 = 0.22, 2 = 0.33 and 2-1 = 0.44. Seminal stains kept for more than 6 months revealed distinguishable band patterns as well as fresh samples.     

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.     

Population data of casework in West Virginia on six genetic marker systems

Blood specimens and stains submitted from all geographic regions of West Virginia were analyzed for six genetic markers: International ABO, phosphoglucomutase (PGM), esterase D (ESD), erythrocyte acid phosphatase (EAP), adenylate kinase (AK), and adenosine deaminase (ADA). The four-year study indicates that markers identified were distributed in Hardy-Weinberg equilibrium and are consistent with population data previously reported.     

复合扩增mtDNA-HVI和Cyt b片段鉴定混合血痕种属

目的探讨mtDNA-HVI和Cyt b片段复合扩增法鉴定人与动物混合血痕种属的应用价值。方法用chelex-100法从人、牛、猪、狗、兔、鱼、鸡和鼠血痕中提取DNA,复合扩增mtDNA-HVI片段和Cyt b片段,琼脂糖凝胶电泳检测。结果人类在mtDNA-HVI区和Cyt b区分别出现279bp和358bp各一条带,且279bp条带亮于358bp;动物均只有358bp一条带。人与7种动物血痕的检测灵敏度均为3.13ng。检测人与动物混合DNA,灵敏度仍为3.13ng,但358bp条带亮于279bp条带。结论当358bp带明显强于279bp带时,提示检材为人与动物的混合。     

Immunological detection of human phosphoglucomutase (PGM 1) subtypes

Anti-phosphoglucomutase (PGM) antibodies have been produced by immunising a sheep with a purified preparation of rabbit skeletal muscle PGM and used to devise an immunological procedure for detecting PGM isozymes after isoelectric focusing. The anti-rabbit PGM antibodies cross react with human PGM and can be used to identify the PGM1 isozymes characteristic of this polymorphism. The patterns revealed by immunodetection are exactly comparable with those obtained by isozyme staining.     

Hemolyzed blood and serum levels of delta 9-THC: effects on the performance of roadside sobriety tests

A pilot study was conducted to ascertain the range of induced hemolyzed blood/serum delta 9-tetrahydrocannabinol (delta 9-THC) concentrations in 58 human subjects. Subjects were tested within 5 min of smoking a delta 9-THC cigarette and then at half-hour intervals to 150 min. The subjects initially demonstrated a broad range of delta 9-THC hemolyzed blood levels, which settled within an hour to levels comparable to those measured in California drivers who had been stopped for impaired driving, arrested, and tested for delta 9-THC. Serum levels, when correlated with performance or roadside sobriety tests, demonstrated a broad range (5 to 183 ng/mL) of delta 9-THC levels and an "adaptation" effect in the subjects' perception of their own impairment. Although this preliminary study was not a double-blind placebo experiment, the overall performance of human subjects demonstrated the "adaptation" effect, which may be a significant factor in making judgments while performing such complex tasks as driving. Also, the effects of the drug extended beyond the period of elevated delta 9-THC blood levels, perhaps because of THC metabolites that may contribute to impairment or the persistence of THC in the central nervous system. This pilot study will lay the groundwork for a program designed to determine the epidemiology and behavior correlates of marijuana use in motorists.