Identification of novel genes expressed in hypoxic brain condition by fluorescence differential display

Fluorescence differential display (FDD) and comparative RT-PCR have been used extensively to detect differentially expressed genes. We investigated hypoxia-induced gene expression in the brain by FDD-PCR and comparative RT-PCR. Mice were anaesthetized after which hypoxia was induced by neck ligation for 1 min or 25 min, then were killed by decapitation, and the brains were dissected either immediately or 30 min after death (Group A1-0, Group A25-0, Group A1-30 and Group A25-30). Control mice without ligation of the neck were killed by decapitation under anaesthesia immediately (Group C-0) or 30 min after death (Group C-30). FDD-PCR, sequence analysis and comparative RT-PCR revealed that mitochondrial thymidine kinase 2, Rab6, selenoprotein T and two novel cDNAs were enhanced in Group A25-0 and Group A25-30 compared with the other groups. In Group A25-30, only RAP2 interacting protein and another novel cDNA were induced. In Group A25-0, one novel gene was detected. These findings were consistent with the results of statistical analysis by ANOVA. No differences of band pattern were observed among Groups A1-0, A1-30, C-0 and C-30. The genes exhibiting altered expression were associated with vital cellular functions, e.g., intracellular signaling and mitochondrial metabolism. In addition, we identified four novel genes, expressed after extended hypoxic conditions in mouse brain with suffocation. These results may contribute to clarify the pathophysiology of asphyxia in the brain and aid in the diagnosis of suffocation.     

中国汉族群体人类补体C8A多态性

采用免疫沉淀、SDS-聚丙烯酰胺凝胶电泳 (SDS- PAGE)、被动转印及酶免分析 ,研究了人类补体 C8A等位基因频率在成都地区汉族群体中的分布。 12 1份样本被分为 3种常见型 ,即 C8A- A、C8A- B及 C8A- AB,由两个等位共显性基因 C8A * A及 C8A* B控制 ;同时发现了 2个稀有亚型 ,即 A3亚型及新发现的 Ax亚型。等位基因频率为 C8A* A=0 .5 0 83,C8A* B=0 .4835 ,C8A*稀有型 =0 ,0 0 83。说明 C8A多态性在中国群体中具有良好的分布 ,个人识别率(DP)达到 6 1.14% ,可用于法医学个人识别及亲子鉴定     

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.     

Analysis of Dopamine D2 Receptor (DRD2) Gene Polymorphisms in Cannabinoid Addicts*

Abstract: The gene encoding the dopamine D2 receptor (DRD2) has been suggested as a candidate gene for substance dependence. In this study, the possible association between Taq1A and Taq1B DRD2 polymorphisms and cannabinoid dependence was investigated. One hundred and twelve cannabinoid addicted and 130 healthy control subjects were included in this study. The Taq1A and Taq1B genotypes were determined in all subjects by polymerase chain reaction. For each polymorphism (A or B), the subjects were categorized into three groups according to their genotype, that is, the subjects with alleles A1/A1, A1/A2, A2/A2; B1/B1, B1/B2, and B2/B2. A significant association was found between Taq1A gene polymorphism and cannabinoid addicts compared to the control subjects. This finding suggests that polymorphism of the Taq1A, but not the Taq1B, may be associated with the susceptibility to cannabinoid dependence. Further clinical studies are required to be carried out for confirmation and evaluation of these findings.     

Y染色体3个SNP基因座及其单倍型的遗传多态性和群体差异

目的 探讨人类Y染色体3个SNP基因座及其单倍型的遗传多态性和群体差异。方法 应用PCR-RFLPs结合DNA序列分析技术,对140例来自中国藏族、日本、南非黑人及南非白人男性的Y染色体M4、M9和M122基因座的等位基因进行分析。结果 全部样品M4基因座的等位基因均为野生型M4A,未发现多态性。共检出3种单倍型,黑人个体均为野生型单倍型M4A/M9C/M122T。白人个体有8例单倍型为M4A/M9G/M122T,未检出等位基因M122C。日本及中国藏族群体以单倍型M4A/M9C/M122T为主,频率分别为0.50和0.65,未检出单倍型M4A/M9C/M122C,个人识别机率与父权排除率分别为0.6191和0.4994。单倍型频率分布在中国藏族和日本群体之间存在显著性差异(P<0.01)。结论 单倍型M4A/M9G/M122C为亚洲人特征,M9和M122基因座在中国藏族和日本群体中具有较高的遗传多态性,并显示出明显的人种和群体差异。     

Analysis of hallucinogenic constituents in Amanita mushrooms circulated in Japan

The constituents of seven mushrooms sold as Amanita muscaria or Amanita pantherina (five A. muscaria and two A. pantherina) and four "extracts purported to contain A. muscaria" products that are currently circulated in Japan were determined. All mushroom samples were identified as A. muscaria or A. pantherina by macroscopic and microscopic observation. The dissociative constituents, ibotenic acid (IBO) and muscimol (MUS), were extracted with 70% methanol twice and determined by gas chromatography/mass spectrometry. The IBO (as the hydrate)/MUS contents were in the range of <10-2845ppm/46-1052ppm in the cap of A. muscaria and 188-269ppm/1554-1880ppm in the cap of A. pantherina. In the caps, these compounds had a tendency to be more concentrated in the flesh than in the cuticle. On the other hand, the IBO/MUS contents in the stem were far lower than in the caps. In the "extracts purported to contain A. muscaria" products, IBO/MUS were detected below the lower limit of calibration curve (<10ppm/<25ppm) or not detected. However, these samples contained other psychoactive compounds, such as psychoactive tryptamines (5-methoxy-N,N-diisopropyltryptamine and 5-methoxy-N,N-dimethyltryptamine), reversible monoamine oxidase inhibitors (harmine and harmaline) and tropane alkaloids (atropine and scopolamine), which were not quantified. This is the first report of the chemical analysis of Amanita mushrooms that are circulated in the drug market.     

A Spanish population study of 17 Y-chromosome STR loci

Haplotype, allele frequencies and population data of 17 Y-chromosome STR loci DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS460 (GATA A7.1), DYS461 (GATA A7.2), GATA A10, GATA C4 and GATA H4 were determined from a sample of 148 unrelated male individuals from Spain. A total of 144 haplotypes were identified by the 17 Y-STR markers, of which 141 were unique, two were found in two individuals and one was found in three individuals. The haplotype diversity (99.95%) and discrimination capacity (97.30%) were calculated. Comparisons were made with previously published haplotype data on other Iberian population samples and no significant differences were found.     

HLA—A基因座分步PCR—SSP分型研究

目的使HLA基因分型能应用于法医常见检材的个人识别。方法 建立检测HLA—A基因座的分步PCR—SSP方法。先用一对HLA—A基因座特异的引物作第一次扩增,以所得产物为模板,分别用对HLA—A30、A31、A33特异的3对引物作第二次扩增,二次扩增的产物经电泳判型。结果 1130例血清分型为HLA—A30、A31、A33的血痕,其PCR—SSP分型和血清分型的不符合率为29％;室温保存2年的精斑、唾液斑,保存18年的血痕第一次扩增均获得满意的结果。结论法医亲子鉴定和个人识别宜用基因分型替代血清分型。HLA—A基因座分步PCR—SSP基因分型适用于法医检材。     

FUT2基因座4个新变异等位基因的分析

目的 探讨FUT2基因座新变异等位基因的结构、检测方法与表达状态。方法 应用PCR、RFLPs、基因重组、DNA序列检测及基因表达技术,对4种FUT2基因座新变异等位基因进行分析。结果在新几内亚人个体中,发现了3种新的FUT2等位基因,分别由错义突变C664T、G868A和G760A所致。经基因表达证实:3种基因编码的α2-FUT酶蛋白缺乏相应的糖基转移酶活性;在中国汉族个体中,发现1例同义突变A660T。C664T和A660T改变了限制性内切酶Sac Ⅰ的识别序列,可以用RFLPs方法进行检测。在应用DNA序列分析技术检测杂合子时,可能会漏检显示弱峰的变异,RFLPs技术不能确定内切酶识别序列内部的具体变异点。结论　 C664T、G868A和G760A突变所形成的FUT2基因为非分泌型基因,序列多态性的检测应使用2种以上的方法相互验证。     

Urinary catecholamines in hyperthermia-related deaths

A group of five hyperthermia-related deaths is presented in which urinary noradrenaline (NA) concentrations were elevated (172.1 +/- 119.4 ng/ml) compared with a control group of rapid violent deaths (43.7 +/- 26.1 ng/ml). Urinary adrenaline (A) concentrations were not elevated in the hyperthermia cases, nor were there any significant differences in urinary dopamine (DA) concentrations between the two groups. All except one of the hyperthermia victims were under the influence of ethanol. It is suggested that a combination of heat stress and ethanol consumption was responsible for the elevated urinary NA in the hyperthermia cases, reflecting increased sympathetic nervous system activity. A combination of high urinary NA with low A seems to be characteristic of hyperthermia fatalities, in contrast to hypothermia deaths, where both NA and A are usually elevated.     

家兔死后心肌酰胺A带的傅里叶变换红外光谱图像分析

目的 应用傅里叶变换红外光谱成像系统研究家兔心肌酰胺A带变化与死亡时间的关系.方法 将32只家兔处死后取出心脏,20℃保存,于48h内不同时间点取样并制作切片.用傅里叶变换红外光谱成像系统绘制酰胺A带图像,分析研究死后酰胺A带阳性与阴性面积比的变化规律. 结果 48h内随死亡时间(x)的延长,酰胺A带的阳性与阴性面积比...     

An unusual case of identification by DNA analysis of siblings

A badly decomposed body required identification by means of DNA analysis. A brother and sister of the deceased were available as reference subjects. Although investigation of Y-chromosomal markers established an exclusion condition, autosomal markers suggested a positive identification. In order to increase the reliability of the tests, X-chromosomal markers were also investigated. This analysis showed the body to have an XXY genotype (Klinefelter's syndrome). A number of hypotheses were assessed using biostatistical methods, ultimately resulting in a definite identification. The special aspect of Klinefelter's syndrome proved highly useful for biostatistical analysis.     

Development of a DNA-based macroarray for the detection and identification of Amanita species

A DNA-based macroarray was designed to quickly and accurately identify certain Amanita mushroom specimens at the species level. The macroarray included probes for Amanita phalloides and Amanita ocreata, toxic species responsible for most mushroom poisonings, and Amanita lanei and Amanita velosa, edible species sometimes confused with toxic species, based on sequences of the highly variable internal transcribed spacer (ITS) region of rDNA. A cryptic species related to A. ocreata and one related to A. lanei, identifiable by ITS sequences, were also included. Specific multiple oligonucleotide probes were spotted onto nylon membranes and the optimal hybridization temperatures were determined. The Amanita DNA array was highly specific, sensitive (0.5 ng DNA/μL and higher were detected), and reproducible. In two case studies, the method proved useful when only small amounts of mushroom tissue remained after a suspected poisoning. An identification could be completed in 12 h.     

共有基因座数和等位基因数用于结直肠癌组织的身源认定

目的 探讨结直肠癌组织中STR基因座变异情况及其身源认定方法. 方法 用Identifiler系统对50对新鲜结直肠癌组织及其身源正常组织(CR-N)组进行STR分型,计算CR-N组中变异STR基因座及全不同基因座数(A0)、半相同基因座数(A1)、全相同基因座数(A2)和共有等位基因数(IAn),比较CR-N组、无关个体对(UI)组和全同胞对(FS)组中上述参数的分布差异,通过判别分析建立判别函数.结果 结直肠癌组织中STR基因座基因型改变发生率为3.33%.CR-N组中A1、A2和IAn呈显著偏态分布并与其在UI或FS组中分布差异显著.依据IAn、A1/A2分别建立了CR-N与UI、CR-N与FS的判别函数,其对结直肠癌组织身源认定错判率均为0.00%. 结论 结直肠癌组织中STR基因座基因型改变发生率较高;本研究所建立的判别函数是进行结直肠癌组织身源认定的一种可行方法.     

Sequence analysis of STR polymorphisms at locus ACTBP2 in the Taiwanese population

A highly polymorphic sequence structure is reported in the human beta-actin related pseudogene 2 (ACTBP2) (SE33) locus in members of the Taiwanese Han population. A total of 100 unrelated members of the Taiwanese Han population were used in the study. Alleles that shared the same size but differ in their sequence are described to allow for inter laboratory sharing of data. PCR products amplified from this locus were separated by single-strand conformation polymorphism electrophoresis, the single-stranded DNA bands were excised from the gels, a second amplification performed, and then the PCR products were sequenced. All the alleles differed by either 2 or 4 bp. Sequence variations were observed as deletions or insertions in the repeat units AG (or AA) and AAAG. Additionally, transitions in the flanking regions were recorded. A total of 27 alleles with 71 associated genotypes were recorded if the alleles were defined by size, but 68 alleles with 88 associated genotypes were noted with the alleles were scored on the basis of sequence variation. The power of discrimination (Pd) of this single locus was 0.9874 making the human ACTBP2 a good alternative marker for individual identification and paternity testing.     

多重PCR检测FFv三个基因座在景颇族人群中的遗传多态性

短串联重复序列（STR）是由几个碱基对作为核心单位串联重复形成的一类DNA序列,作者将3个STR基因座在同一反应体系中进行互不干扰的复合扩增,采用高分辨率的聚丙烯酰胺凝胶电泳分离、银染法显影技术,对云南省景颇族的F13A01,FESFPS和vWA等3个基因座等位基因的基因频率进行了调查,获得了满意的结果,显示了广阔的应用前景。F13A01基因座观察到8个等位基因、13个基因型;FESFPS基因应观察到7个等位基因、18个基因型;vWA基因应观察到7个等位基因、21个基因型。     

23例头面部拳掌伤致脑干损伤致死病理学分析

目的 观察头面部拳掌伤，及钝器打击伤致脑干损伤死的病理形态学特点，并探讨其成伤机理。方法 对23例拳击(A组)与30例钝器打击(B组)采用作者建立的脑干损伤取材方法，于脑干各颅神经根部作水平面切6块，HE染色光镜检查。结果 2组脑干损伤具有相同的病理形态学改变。A组水肿显著(71.6％)，B组以组织撕裂、挫碎为多(76.6％)；A组颅神经损伤多为单根(47.6％)，B组2根以上占53.7％，最多可达4根；A组单纯脑实质挫伤多(50％)，B组多伴发血肿或挫挤带。结论 拳、掌和钝器打击头面部致脑干损伤的病理形态学相同，但钝器打击比拳、掌致脑干损伤严重且广泛。提示脑干损伤致命与其受力部位、发病机理关系密切，而与受力强度关系不大。     

A quantitative differentiation method for acrylic fibers by infrared spectroscopy

Absorbance peak areas of nitrile (2240 cm(-1)), carbonyl (1730 cm(-1)) and CH (1370 cm(-1)) groups were obtained for 48 colorless acrylic fibers by infrared (IR) microspectroscopy. The carbonyl signal, related to the comonomers most commonly used in acrylic fibers, was ratioed against the nitrile and CH bands, pertaining to the backbone of the polymer chains. The ratios A1730/A2240 and A1730/A1370, a relative measure of the comonomer content in the fiber, were used to differentiate the samples. A decrease in the crystallinity of fibers has been noted with increasing comonomer content. Relative standard deviation (R.S.D.) of the ratios were 1 and 3% for repetitive analyses on the same location and along the length of the same single fiber, respectively. When different fibers of the same sample were examined, results were reproducible within 6%. This simple method can greatly enhance the evidential value of colorless acrylic fibers, being able to discriminate them and thus helping the Court to better assess their significance.     

Population data of new Y-chromosome STRs GATA C4, DYS438, DYS437, GATA A7.2, GATA H4, DYS439 and GATA A10 in males from Colombia

Two hundred twenty-five unrelated males were typed for 7 over 8 loci Y-chromosome STRs proposed in a collaborative study by The Spanish and Portuguese ISFG Working Group. The markers amplification were in two multiplex reactions GEPY I with GATA C4, DYS438, DYS437, DYS461 (GATA A7.2) and GEPY II with GATA H4, DYS439, GATA A10 and DYS460 (GATA A7.1). All gene diversities were upper 0.5 with the highest value in DYS439 with 0.64. Furthermore, 152 haplotypes from 7 loci Y-chromosome STRs were found within studied population and a high haplotype diversity 0.9902 was found. The DYS460 (GATA A7.1) marker can not be studied because its diverse alleles were not able for interpret.     

Population study of HUMTH01, HUMVWA31/A, HUMF13A1, and HUMFES/FPS systems in Azores

The tetrameric short tandem repeat polymorphisms HUMTH01, HUMVWA31/A, HUMF13A1, and HUMFES/FPS were studied in blood stains obtained from a population of unrelated individuals from the Azores Archipelago (Portugal). Gene frequencies were determined and no deviation from the Hardy-Weinberg equilibrium was found. However, the allelic independence test between loci showed linkage disequilibrium between HUMVWA31/A and HUMFES/FPS. A combined discrimination power and chance of exclusion of, respectively, 0.9999 and 0.9534, reveal the high forensic interest of the four systems. No differences with other caucasoid populations were found, but comparison with some asiatic, eskimo, and amerindian populations showed significant statistical differences.