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.     

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.     

The forensic identification of semen by isoelectric focusing of seminal acid phosphatase.

An isoelectric focusing technique for the separation of acid phosphatases is described. The focusing patterns for semen and vaginal secretion are examined in detail and the technique is evaluated as a means of identifying and distinguishing between these body fluids and other materials of potential forensic interest. The method can reliably be used to diagnose the presence of semen in stains and in the post-coital vagina.     

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 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.     

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.     

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.     

Double replica electroblotting for the simultaneous phenotyping of C6 and C7, and its application to the examination of bloodstains

A new reliable and reproducible technique for the simultaneous determination of C6 and C7 types is presented, which employs double replica electroblotting after isoelectric focusing. It permitted clear discrimination of both C6 and C7 components, and the patterns were nearly comparable to those demonstrated separately. The population data obtained by this new technique fitted the genetic hypothesis. The present double replica electroblotting method was successfully applied to the combined phenotyping of C6 and C7 from bloodstains which were stored at room temperature for up to 4 weeks. The method is quite suitable for medicolegal examination of bloodstains particularly for the saving in the amount of sample.     

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.     

Lactate dehydrogenase isozymes in vaginal swab extracts: A problem for the identification of menstrual blood

Extracts of vaginal swabs free of both blood and semen, collected from three donors throughout several menstrual cycles, have been examined by cellulose acetate membrane electrophoresis for the presence of lactate dehydrogenase (LDH) isozymes. LDH activity, predominantly in the form of LDH-5 was detected in some extracts. Mixtures of these particular extracts with peripheral blood gave LDH patterns similar to those observed for menstrual blood. The results indicate that LDH isozyme patterns cannot be used to differentiate between stains of menstrual blood and bloodstains that have become mixed or contaminated with vaginal secretions.     

Haptoglobin phenotyping by polyacrylamide gel isoelectric focusing and its application to simultaneous typing of serum proteins

A simple isoelectric focusing method for haptoglobin (HP) typing is described. Serum was pretreated first with C. perfringens neuraminidase (CPN) and then with dithiothreitol (DTT). The treated serum was subjected to polyacrylamide gel isoelectric focusing (PAGIF), and the band patterns were detected by immunoblotting. The method could be successfully applied to HP typing of bloodstains as old as 2 months. A slight modification of it enabled HP, complement component C81, and factor I (IF) to be typed simultaneously. The immunoblotting facilitated preservation of HP patterns. Thus, the PAGIF method for HP typing is suitable for routine use in the forensic laboratory.     

A method to increase the volume of sample applied to isoelectric focusing gels

Isoelectric focusing of extracts from diluted or aged bloodstains may be more successfully accomplished with larger sample volumes applied to the gel. A technique is described using teflon tubing to apply larger sample volumes (up to 100 μl) to isoelectric focusing (IEF) gels. This method is reproducible and easy to perform.     

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。     

经济发展方式转变的数理逻辑与系统思考

本文围绕加快转变经济发展方式是我国当前经济工作的紧迫任务、科技进步促进经济发展方式转变的历史回顾、科技创新优化经济发展方式的现实比较、经济发展方式转变的数理逻辑与阶段特征和加快我国区域经济发展方式转变的路径选择等方面对转变经济发展方式问题进行了分析,并探讨了我国当前区域经济发展的阶段性特征及其发展规律。     

Group specific component subtyping in bloodstains by separator isoelectric focusing in micro-ultrathin polyacrylamide gels followed by immunoblotting

The identification of group specific component (Gc) subtypes derived from blood-stains by separator isoelectric focusing in micro-ultrathin polyacrylamide gels (interelectrode distance: 50 mm) containing 4.5 to 5.4 pharmalytes is described. The separation achieved between Gc 1F and Gc 1S bands is compared favorably with that obtained using separator isoelectric focusing in conventional polyacrylamide gels dimensions (interelectrode distance: 110 to 120 mm). The technique is rapid and economical, and the immunoblotting method described is more sensitive than immunofixation followed by silver staining.     

A hybrid ampholyte focusing technique for esterase D subtyping of evidentiary material

An ultrathin-layer polyacrylamide gel isoelectric focusing technique that uses a composite of ampholytes from three commercial sources is described for subtyping esterase D. All common allelic products of esterase D were separated clearly. The technique described in this paper provides a higher conclusive call rate on known blood specimens (95.8%) and questioned bloodstains (69.7%) compared with continuous zone electrophoresis in agarose gels (89.9 and 37.6%, respectively).     

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.     

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.     

Group specific component: isoelectric focusing subtyping and immunoblot detection

A method for the detection of group specific component (Gc) by immunoblotting, following isoelectric focusing (IEF), is described. This isoelectric focusing method resolves the six common phenotypes of Gc using a narrow range pH 4.5 to 5.4 ampholyte. The Gc proteins were passively transferred from the IEF gel to nitrocellulose and detected with goat anti-Gc followed by peroxidase labeled anti-goat immunoglobulin (Ig) antibody. The increased sensitivity of this technique results in the typing of stains older than one year and also those stains with minimal concentrations of the Gc protein. The polyacrylamide gel can also be used for the subtyping of esterase D.