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.     

PGD phenotyping in bloodstains, organ tissues, dental pulps and hair roots by isoelectric focusing

Polymorphism of PGD was investigated in bloodstains, organ tissues, dental pulps, hair roots and semen by isoelectric focusing. This technique provided much higher resolution of PGD isoenzymes than starch gel electrophoresis. Phenotyping was possible from bloodstains for 5 weeks, from organ tissues (except pancreas) for 1-3 weeks, from dental pulps for 2 weeks and from hair roots for 2 weeks when they were stored at room temperature. The method is simple, rapid, reliable and therefore useful in medicolegal individualization of bloodstains, organ tissues, teeth and hairs.     

人体组织黄递酶Ⅲ分型

应用超薄层PAGIEF对人体组织作DIA3分型，结果显示睾丸、卵巢、子宫肌和牙髓组织均可检出DIA3多型带。脑、肝、肾、肾上腺、脾、肠、胰腺、心肌、肺、皮肤和骨骼肌以及血液、阴道液和毛根均未检测出DIA3。应用紫外分光光度法测定人体不同组织，血液和阴道液中DIA相对活性，结果各组织DIA活性相对值与PAGIEF中DIA显色谱带密度基本一致。作者认为，用PAGIEF对生殖器官和牙髓进行DIA3分型在法医的个人识别中有一定意义，在混合斑鉴定中可以不考虑阴道液对精液DIA3分型的干扰。     

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.     

Typing study of human semen DIA3 by isoelectric focusing--distribution in the Wuhan population, China

Human semen DIA3 typing was studied by isoelectric focusing on ultra-thin-layer polyacrylamide gel which resulted in a simpler and more definite separation of the products of DIA3 alleles than hitherto. In 198 semen samples collected from unrelated Chinese males four different phenotypes were observed. The DIA3 allele frequencies were calculated: DIA 3(1) = 0.7727, DIA 3(2) = 0.2172, DIA 3(3) = 0.0101. The results of the stability study of 12 laboratory-prepared semen stains stored at room temperature suggested that DIA3 in seminal strains is a relatively stable genetic marker. Our gene frequencies have been compared to those reported in other populations.     

Esterase D phenotyping of bloodstains and hair roots by low voltage isoelectric focusing

A new isoelectric focusing method is described for phenotyping of esterase D in blood stains and hair roots. It permitted easy and rapid discrimination of six phenotypes determined by ESD*1, ESD*2 and ESD*7. Experiments showed it to be practicable in forensic stain work. In addition, this technique was also usable in phenotyping of ESD 5.     

alpha-L-fucosidase phenotyping in human tissues, dental pulps and hair roots

Phenotypes of alpha-L-fucosidase (Fu) were demonstrated from tissue samples of spleen, liver, lung and kidney stored for a few weeks at room temperature. Activity was fairly low in pancreas, heart, muscle, skin and brain, and typing was not reliable after 1 week storage. Fu types were detectable from dental pulp tissue of up to 1 week storage. Activity was present in hair root cells, but was extremely low. The results show that the Fu typing may be applicable to individual identification of organ tissues and teeth.     

Use of the isoelectric focusing method in examining hair proteins

Method of isoelectric focusing in thin and ultrathin layer of polyacrylamide gel followed by staining the polypeptide fractions with argentum was used to investigate human hair keratins. Method makes it possible to determine the polymorphism of keratins in hair even 1,5-2 cm long. It helps to obtain the additional information in medicolegal expertise of hair affinity.     

The isoelectric focusing of keratins in hair followed by silver staining

An isoelectric focusing method followed by silver staining has been developed for the study of keratins which is as effective as two-dimensional electrophoresis and fluorography for hair species identification. Hair from dogs, rabbits, horses, cows, guinea-pigs, donkeys, sheep and cats were successfully identified. Narrow pH ranges were used to observe heterogeneity in human hair. Although this heterogeneity may be affected by environmental conditions, it may be of use in criminalistics.     

An improved means of enzyme typing of hair roots using isoelectric focusing.

An improved method of grouping hair, based on the alleles of PGM observed by isoelectric focusing, has been described. The increased discriminating power of this system (0.77) compared to that obtained by the starch gel technique (0.55) provides a new and more sensitive means of typing hair.     

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.     

人与动物毛发角蛋白组分的等电聚焦图谱分析

本文用聚丙烯酰胺等电聚焦(PAGIEF)对来自6个目、15个科、20种动物毛发角蛋白组分进行了研究。发现人与动物毛发角蛋白组分的PAGIEF谱型有显著差异;不同种属的动物毛发角蛋白组分的PAGIEF谱型也互不相同,它们各自都有独特的角蛋白PAGIEF谱型。作者认为,本方法可用于鉴定毛发的种属。     

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.     

A method of human diaphorase locus 3 phenotyping in human semen by isoelectric focusing

A rapid method for the separation of diaphorase (DIA 3) isozymes in the pH range 6-8 is presented. Isoelectric focusing (IEF) of DIA 3 on 0.2 mm thick polyacrylamide gels took 60 min at 5 degrees C. Separated bands are sharp and common phenotypes are readily distinguishable. The method combines high resolution, speed, reproducibility and reagent economy with simplicity.     

Electrophoretic analysis of non-human primates hair keratin

Keratin characterization through electrophoretic techniques has been used for species identification in forensic science and in taxonomic studies. In the present work, protein components solubilized from hair of non-human primates were evaluated to investigate whether there is any species-specific pattern in an evolutionary perspective, by grossly comparing hair native keratins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Extracted hair keratins for all specimens were separated by SDS-PAGE into 2–3 polypeptide bands with apparent MW in the range 39–54 kDa and into 3–7 polypeptide bands with apparent MW in the range 10–35 kDa. With this technique it was possible to distinguish different suborders, different families of the same suborder, and, sometimes, different genera from the same family. On the contrary, it was not possible to distinguish different species of the same genus and different specimens of the same species. With IEF, extracted hair keratins were separated into about 30 polypeptide bands with pI values in the range of pH 3.9–7.7. IEF discriminates poorly between different samples. Only in specimens from Papio genus did we find an additional polypeptide band. In conclusion, we found that the differences between electrophoretic patterns are largest for animals that are not closely related while specimens of the same species have the same patterns.     

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.     

Transferrin subtyping of human bloodstains

A method was described for subtyping transferrin derived from human bloodstains. Bloodstain cuttings were extracted in 0.5% ferrous ammonium sulfate. The extracts were subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. After isoelectric focusing, transferrin was detected by silver staining. This method permitted the successful typing of Tf in 6-month-old blood stains maintained at -20 degrees C and room temperature and 3-month-old bloodstains maintained at 37 degrees C.     

Possibilities of DNA sex determination in hair roots

Species- and sex-determination on hair roots were simultaneously performed using extracted DNA and a human Y chromosomespecific probe. (pH Y 2.1) After Southern hybridization, Hae III-digested DNA fragments were detected by non-radioactive digoxigenin detection system. DNA was extracted from one to five freshly plucked hair roots. The specific 2.12 kb fragment was successfully detected in male DNA samples from a single hair root. A positive identification of female DNA was more difficult. The hair root DNA was revealed to be stable at room temperature for at least 2 weeks (examination time) and produced the same specific band pattern as the DNA of fresh hair roots. In the blind tests with DNA samples from randomly plucked one to four hair roots, the rate of successful sex-determination was 95.8% on male samples (23 out of 24 samples) and 25% on female samples (4 out of 16 samples).     

The ultrastructure of tissue attached to telogen hair roots

Most tissues encountered in forensic biology laboratories have been previously characterized with electron microscopy due to their medical importance. Anagen hair root cells, epithelial cells, erythrocytes, neutrophils, osteocytes, and spermatozoa have received considerable research attention at the ultrastructural level. There is no literature indicating that cells attached to removed telogen hair roots have been characterized with transmission electron microscopy. Nonetheless, telogen hairs are a frequent submission item to forensic laboratories for DNA typing. The amount of tissue attached to a telogen hair root usually determines whether that hair is suitable for nuclear DNA typing methods or mitochondrial DNA typing methods. This study used transmission and scanning electron microscopy to characterize the tissues found in three commonly occurring telogen hair root forms. The tissues were found to consist of keratinized remnant follicle, nonnucleated epithelial cells, nucleated epithelial cells, and trichilemmal keratin. These findings were consistent with the known principles of hair follicle regression. The recognition of the root structures that contain these specific tissue types may assist in the DNA typing of telogen hairs inasmuch as the quality of tissue present may be more important than the amounts of tissue present.     

Subtyping phosphoglucomutase-1 in semen stains and bloodstains: a report on the method

A method is described for obtaining nondistorted, reproducible phosphoglucomutase-1 subtyping patterns from semen stains and bloodstains. Isoelectric focusing of phosphoglucomutase-1 was accomplished in 80 min in a 0.2-mm-thick polyacrylamide gel with an interelectrode wick distance of 8.0 cm. The gel contained 1.2% (w/v) N-(2-hydroxyethyl) piperazine-N-3-propanesulfonic acid (EPPS) and pH 5 to 7 ampholytes (4% w/v). When maintained at room temperature, laboratory-prepared bloodstains and semen stains could be typed for phosphoglucomutase-1 up to four months and three weeks, respectively. An evaluation of phosphoglucomutase-1 typing by isoelectric focusing and the Group I system was performed on casework samples submitted to the FBI Laboratory. In addition to the increased discriminating probability of phosphoglucomutase-1 when subtyped, isoelectric focusing yielded an increase in positive calls on questioned bloodstains (65.6 versus 36.2%) and dried seminal stains (16.4 versus 13.1%) compared with the Group I system.