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.     

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。     

应用等电聚焦电泳对人精液黄递酶Ⅲ表型分布的研究

应用超薄层聚丙烯酰胺凝胶等电聚焦电泳对武汉地区198例成人精液黄递酶Ⅲ(DIA_3)作了分型。观察到4种表现型,基因频率分别为DIA_3~1=0.7727;DIA_3~2=0.2172;DIA_3~3=0.0101。对保存精斑进行检测证实,室温下保存9周的精斑仍能作DIA_3分型。在聚丙烯酰胺凝胶中引入分离剂HEPES,提高了分辨率。改进后的等电聚焦技术尤适于DIA_3基因产物的分离。文中还调查了我国汉族人群DIA_3频率分布,并与其它群体资料作了比较。     

alpha-L-fucosidase phenotyping in human placentae, semen and seminal stains

The polymorphism of alpha-L-fucosidase (Fu) was investigated in a Japanese population from samples of placentae and semen, using isoelectric focusing. The gene frequencies of placental types were Fu1 = 0.748 and Fu2 = 0.252, and those of seminal types were Fu1 = 0.739 and Fu2 = 0.261. The coincidence in the distribution between the placental and seminal types suggests that the Fu types occurring in placentae and in semen are controlled by the same Fu alleles. The Fu typing was possible in seminal stains stored at 4 degrees C for up to 9 weeks, at room temperature for up to 7 weeks and at 37 degrees C for up to 4 weeks. The Fu types were still detectable at semen dilutions of up to 1:4. This polymorphism would provide a useful genetic marker for the medicolegal grouping of seminal stains.     

精浆r—谷氨酰转移酶多态性研究

本次实验采用聚丙烯酰胺阶段梯度凝胶电泳方法,对武汉地区135名无关男性精液γ—GT进行了遗传多态性调查,根据γ—GT的电泳差异,分为1型、2型、2—1型三种常见表现型,1型为靠近正极的单一带,2型为靠近负极的单一带,2—1型为1型带和2型带组成的杂合双带。表现型频率分别为0.156,0.356,0.488,基因频率为γ—GT~1=0.400,γ—GT~2=0.600(P>0.5)。室温下保存8周的精斑,也能检测出较清晰的谱带。本次实验还探讨了单纯阴道分泌物及精液、阴道分泌物混合斑进行γ—GT分型的可能性。     

Erythrocyte diaphorases DIA1 and DIA2 in bloodstains

The ability to determine the phenotypes of erythrocyte NADH diaphorase (DIA1) was demonstrated in bloodstains, the utility of the system extending about two weeks. Electrophoretic variants representing three uncommon phenotypes (DIA1 2-1, DIA1 4-1 and DIA1 7-1) were found in five of 785 individuals tested. The NADPH diaphorase DIA2 isozymes could not be resolved sufficiently for practical use.     

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.     

Simultaneous identification of alpha-L-fucosidase and phosphoglucomutase (PGM) subtyping in semen stains

Seminal fluid and stains were analyzed by isoelectric focusing to determine the donor phenotype in the alpha-L-fucosidase (AlFuc) polymorphic system. The enzyme is found in both seminal fluid and spermatazoa. Three common phenotypes exist and can be identified in fluid specimens stored at 4 degrees C for more than a year. Untreated semen specimens display more than eight distinct bands of alpha-L-fucosidase activity with isoelectric points of pH 6.6 and below. Neuraminidase-treated specimens have enhanced banding patterns cathodally with a loss of activity in anodal bands making it easier to phenotype specimens. Semen stains maintained in dehumidified chambers at 25 or 37 degrees C retained activity for at least one month and could be accurately phenotyped. Activity was observed in semen specimens maintained at -20 degrees C in the dried state for a period of one year, whereas a complete loss of activity was observed after two weeks in similar specimens maintained at 25 or 37 degrees C under humid conditions. Of seventy-four semen stains analyzed, two had no apparent activity. Of the remaining seventy-two specimens 56, 32, and 12% were phenotyped as FUC 1-1, FUC 2-1, and FUC 2-2, respectively. Calculated gene frequencies are FUC1 = 0.72 and FUC2 = 0.28. Following analysis of alpha-L-fucosidase, the agarose gel can be chemically developed to reveal the PGM1 subtyping pattern. The ability to phenotype both systems in semen stains significantly improves the ability of the analyst to individualize this type of physical evidence. The probability of discrimination for these two combined systems is approximately 0.89.     

中国人精子黄递酶Ⅲ型的分布及精斑黄递酶Ⅲ型的检出

应用超薄层聚丙烯酰胺凝胶等电聚焦技术对辽宁地区200例正常人精子黄递酶Ⅲ型(DIA)的分布进行了研究。其基因频率分别为DIA_3~1=0.6850,DIA_3~2=0.2875,DIA_5~3=0.0275。由阴道液、唾液、乳汁及无精子症精液不能判定DIA_3型。室温保存精斑可检出时限为9个月。     

DIA3 phenotyping in human tissues, dental pulps, and hair roots by isoelectric focusing

The polymorphism of DIA3 was investigated in tissues of various human organs, dental pulps, and hair roots by isoelectric focusing. DIA3 types were demonstrated from tissues of brain, prostate, testis, ovary, and uterus, but not from tissues of spleen, pancreas, heart, liver, muscle, lung, skin, and kidney. Determination was possible from dental pulps stored at room temperature for up to 2 weeks and from fresh hair roots. The results show that the DIA3 typing by isoelectric focusing is useful for medicolegal individualization of brain, reproductive organs, teeth, and hairs.     

Studies on glyoxalase 1 isoenzymes in semen stains: polymorphism in Himachal (India) population

One hundred and ten pairs of blood and semen samples and their stains were studied to type glyoxalase 1 (GLO 1) isoenzymes using agarose-starch medium. A good agreement was observed between the phenotypes expressed in blood and semen samples of the same donor. No GLO 1 activity however could be demonstrated in the vaginal swabs tested. The gene frequencies of GLO 1 polymorphs in Himachal population has been worked out and their stability studies carried out at -12 degrees C and at room temperature.     

Further alleles of phosphoglucomutase in human semen detected by isoelectric focusing

A technique for identifying further alleles of PGM1 in human semen detected by isoelectric focusing has been described. A survey of 100 semen samples has shown that there was close agreement between the observed and expected gene frequencies of these new alleles on the basis that there were four common alleles determined by the PGM1 locus and not two as originally proposed [1,2]. The use of these new genetic variatns of PGM will considerably enhance the investigation of semen stains in forensic science.     

An improved method for semen alpha-L-fucosidase typing--distribution in the Wuhan population of China

An improved method for the separation of the genetic variants of alpha-L-fucosidase in human semen is described. The method involves, isoelectric focusing in ultrathin-layer PAG containing a mixture of ampholines, pH 5-7 and pH 3.5-9.5, and separator HEPES. The Fu pattern obtained is simple, easy to interpret, and reproducible. The occurrence of fucosidase phenotypes in 189 semen samples from the population of Wuhan was investigated, and the frequencies of the Fu alleles were calculated.     

The evidential value of peptidase A as a semen typing system

Human erythrocyte peptidase A (Pep A) displays a genetic polymorphism in blacks. Its occurrence in human semen was examined for its possible use as a semen typing system. Studies by starch gel electrophoresis, in which the Pep A was located by an improved method, were carried out on semen, semen stains, and vaginal swabs taken at known times after intercourse. In addition, a large number of vaginal swabs, negative for semen, were taken from females throughout their menstrual cycles and examined for Pep A activity. The results indicated that Pep A typing could be carried out on semen and semen stains. However, it was possible to determine the Pep A type on vaginal swabs only when they had been taken within about 3 h after intercourse.     

成都地区汉族群体C_3表型频率调查与血痕中C_3表型的检测

用醋酸纤维素薄膜电泳免疫固定法,调查了成都地区汉族群体 C_3表型频率的分布。在400例无关健康献血员中发现三种 C_3表型,SS 型397例,FS 型2例,SS_(var)型1例,其基因频率是 C_3~S=0.9963,C_3~F=0.0025,C_3~(Svar)=0.0013。表现型观察值与期望值吻合(p>0.95)。37℃和室温中放置2天的血痕,4℃中放置23天的血纱布和放置35天的玻璃上血痕,-20℃中放置至少87天血液的 C_3表型,可全部检出。人血清在室温中放3天,4℃中放13天,-20℃中至少放106天,可以全部检出 C_3表型。5例亲子鉴定案中检查了 C_3系统。     

辽宁汉族人血清类粘蛋白(ORM)型的分布及其在血痕精液(斑)及脑脊液中的检出

本文作者采用PAGIEF结合免疫固定法,调查了辽宁汉族群体(431人)ORM的分布,检出了ORM1型和ORM2型各8种表现型;对不同条件下的血痕标本进行检测,成功地检出了37℃保存2年血痕的ORM1型;首次由脑脊髓液中检出了ORM1型。ORM型的总鉴别机率为0.7043,是进行个人识别和亲子鉴定的良好遗传标记。     

ELISA检测精浆特异蛋白P_(30)鉴定人精斑

<正> 1978年美国Sensabaugh分离出精浆特异蛋白P30,并成功的制备出相应的抗P30血清。1987年我国也研制出抗人精浆特异蛋白P30血清,并应用该种抗血清建立了几种琼脂扩散和电泳方法鉴定人精斑。由于人精液中P30含量不高,平均为1.52±0.676mg/ml,鉴定微量精斑存在困难。为了更有效的提高方法的灵敏度。我们建立了鉴定微量人精斑的ELISA固相P30抗体法,现报告如下。     

Evaluation of four deoxyribonucleic acid (DNA) extraction protocols for DNA yield and variation in restriction fragment length polymorphism (RFLP) sizes under varying gel conditions.

This study originated from discussions and recommendations of the Technical Working Group on DNA Analysis Methods (TWGDAM). Four bloodstain deoxyribonucleic acid (DNA) extraction protocols and five semen stain DNA extraction protocols were evaluated. Nine laboratories participated in the extraction of DNA from 20 bloodstains and 20 semen stains using each protocol. All blood and semen stains originated from a single donor and were prepared under uniform conditions to permit the direct comparison of DNA yields and restriction fragment lengths. The extracted DNA from approximately 600 bloodstains and 700 semen stains was quantified by yield gel analysis and a slot blot hybridization technique. The extracted DNA was digested and restriction fragment length polymorphism (RFLP) patterns were generated using three single-locus probes. The RFLP sizing data produced from the blood and semen stains were evaluated with respect to (1) DNA extraction method, (2) gel length, (3) agarose type, (4) presence or absence of ethidium bromide in the gel, and (5) fragment sizes obtained from DNA isolated directly from the donor's liquid blood. This study demonstrates conclusively that high-molecular-weight DNA can be isolated using either organic or nonorganic DNA extraction protocols and that the resulting RFLP sizes are highly reproducible regardless of gel length, agarose type, or presence/absence of ethidium bromide.     

Gc subtypes determined by ultrathin-layer isoelectric focusing. Distribution in the Veneto population (Italy)

The distribution of Gc phenotypes in the population of Veneto was investigated by ultrathin-layer isoelectric focusing. In our sample (n = 732) the six common phenotypes, Gc 1S, 1F, 1S1F, 2, 2-1S, 2-1F and a further phenotype, GC 1S1C3, were observed and the following frequencies calculated: Gc 1S = 0.560792; GC 1F = 0.159153; Gc2 = 0.277323; Gc 1C3 = 0.002732. Our gene frequencies have been compared with those found in other populations.     

A survey of the concentration of 19-OH F1α/F2α prostaglandins in the semen of fertile, infertile and vasectomized men and their stability in both liquid semen and semen stains

The levels of 19-hydroxy-prostaglandins F₁α/F₂α (PG F) in the semen of 19 vasectomized, 44 infertile and 8 fertile men were determined using a simple RIA technique. The mean concentrations observed in this survey were 45 μg/ml, 49.5 μg/ml and 59 μg/ml, respectively. No significant difference was recorded between the vasectomized and infertile groups; there were too few fertile samples available to undertake a meaningful statistical comparison. No reduction was observed in the levels of this PG in a liquid semen sample retained at room temperature over a 4 week period in the presence of a bacteriostat (sodium azide). However, a 30% reduction in the levels of 19-OH PG F occurred over the same time period when aliquots of the same semen sample were retained at either room temperature or at 4°C without azide. Finally, no reduction was observed in the concentration of 19-OH PG F in a series of 10-μl semen stains stored over a period of 6 weeks at room temperature.