小鼠卵母细胞减数分裂过程中活化Pak1的表达及其与染色体分离的相关性

目的研究活化的Pak1在小鼠卵母细胞减数分裂进程中的表达、亚细胞定位及其与染色体分离的相关性。方法用Western blot方法半定量分析活化的Pak1(~(Ser204)位点磷酸化,pPak1~(Ser204))在小鼠卵母细胞减数分裂各个时期的蛋白表达量;用免疫荧光染色法分析减数分裂进程中pPak1~(Ser204)的亚细胞定位模式及其与纺锤体和微管组织中心(MTOC)的时空相关性。结果 pPak1~(Ser204)在小鼠卵母细胞减数分裂重启时(GVBD)开始表达,其表达量随减数分裂进程逐渐升高,在第一次减数分裂中期(MⅠ)时达到峰值并持续保持至第二次减数分裂中期(MⅡ)。在第一次减数分裂前中期(pro-MⅠ)、MⅠ和MⅡ期,pPak1~(Ser204)与MTOC核心蛋白pericentrin和γ-tubulin共同定位于纺锤体两极,pPak1~(Ser204)同时存在于染色体上;在第一次减数分裂后期(AI)到末期(TelI)进程中pPak1~(Ser204)离开MTOC和染色体,聚集在收缩环部位。结论 pPak1~(Ser204)在卵母细胞减数分裂恢复时开始表达,是一种MTOC相关蛋白,可能通过参与纺锤体结构的形成和维持而调控染色体的分离。     

小鼠卵母细胞减数分裂过程中新型微管蛋白ε-tubulin稳定表达并与纺锤体的组装和维持相关

目的研究新型微管蛋白ε-tubulin在小鼠卵母细胞减数分裂进程中的蛋白表达、亚细胞定位及其与纺锤体的相关性。方法用Western blot法检测ε-tubulin在小鼠卵母细胞减数分裂各个时期的蛋白表达;免疫荧光染色分析ε-tubulin在减数分裂进程中的亚细胞定位模式及其与纺锤体的相关性。结果ε-tubulin在小鼠卵母细胞减数分裂各个时期均有稳定的蛋白表达,在减数分裂前期均匀分布在细胞核中,在生发泡破裂后直到第二次减数分裂中期始终保持与纺锤体微管的共定位特性,并持续存在于染色体上。ε-tubulin对纺锤体毒性药物Taxol和Nocodazole敏感并呈现出与微管一致的反应性。结论小鼠卵母细胞内新型微管蛋白ε-tubulin稳定表达并与纺锤体的组装和维持相关。     

天花粉蛋白对小鼠卵母细胞第1次减数分裂的影响

目的 研究天花粉蛋白（TCS）对小鼠卵母细胞第1次减数分裂的影响。 方法 性成熟昆明雌鼠,注射孕马血清促性腺激素10IU后48h,从卵巢中获取未成熟卵母细胞共480枚,在含不同浓度TCS的培养液中,分组进行体外培养,解剖镜下观察并统计其生发泡破裂（GVBD）率和第一极体（PB1）排出率;免疫荧光染色、激光扫描共焦显微镜分析系统观察各组卵母细胞第1次减数分裂过程中纺锤体形成及染色体排列状况。 结果 TCS浓度≤15mg/L时,卵母细胞GVBD率、PB1排出率及到达第1次减数分裂中期（MⅠ）的卵母细胞百分率无明显变化;TCS浓度为30mg/L和60mg/L时,到达MⅠ期的卵母细胞百分率降低（P<0.05）,纺锤体形成和染色体排列出现异常,PB1排出率低于对照组（P<0.05）;TCS浓度达到120mg/L时,MⅠ期卵母细胞的百分率显著下降（P<0.01）,GVBD率下降（P<0.05）,浓度为240mg/L时明显下降（P＜0.01）。 结论 TCS干扰第1次减数分裂重启及纺锤体形成和染色体排列,阻滞小鼠卵母细胞第1次减数分裂,且这种阻滞作用呈浓度依赖性。     

干细胞条件培养液促进小鼠卵母细胞体外成熟

目的:研究骨髓间充质干细胞(MSCs)条件培养液对小鼠未成熟卵母细胞体外成熟的作用.方法:分离、培养小鼠MSCs,获得MSCs条件培养液.收集3类生发泡期卵母细胞,分别在对照培养基和条件培养液中培养,观察卵母细胞成熟率,判断最佳时间点;FDA、Hoechst33258和PI联合染色评价细胞活力;荧光标记检测皮质颗粒分布、迁移及纺锤体复合物的形成情况.结果:条件培养液组3类生发泡期卵母细胞的成熟率高于对照培养基组;其中,完全/大部分裸露的生发泡卵母细胞和周围有疏松的颗粒细胞包裹的生发泡卵母细胞的最佳体外成熟时间为16h,有完整的数层颗粒细胞紧密包裹的生发泡卵母细胞的最佳体外成熟时间为24 h.体外成熟卵母细胞活力良好,皮质颗粒分布及纺锤体复合物形成与体内成熟卵母细胞一致.结论:MSCs条件培养液有利于小鼠体外成熟卵母细胞核、细胞质同步成熟,提高卵母细胞质量,是一种较好的体外成熟培养体系.     

Akt1和Akt2在小鼠卵母细胞中定位的差别

目的研究小鼠卵母细胞减数分裂恢复过程中Akt1和Akt2在从GV期向GVBD期转化期间亚细胞定位的特点。方法采用间接免疫分析技术观察Akt1和Akt2在小鼠卵母细胞GV期和GVBD期的定位;利用显微注射实验技术将Akt1和Akt2抗体分别注射入卵细胞,在不同时间点观察GVBD的发生的数量。结果 Akt1在小鼠卵母细胞GV期均匀的分布于细胞质,而在GVBD期则分布于细胞膜。Akt2在GV期和GVBD期分布于整个细胞,但是GV期以细胞质更为密集,而在GVBD期以细胞核附近更为密集。结论 Akt1可能参与小鼠卵母细胞减数分裂恢复的调控。     

小鼠生发泡完整卵母细胞一个特异基因的克隆及其在胚胎的表达

目的：克隆和筛选小鼠生发泡完整卵母细胞(Gv卵)特异基因，并检测其一在胚胎中的表达。方法：应用抑制性消减杂交技术(SSH)，建立Gv卵特异的cDNA文库。通过斑点杂交法进一步筛选并对阳性克隆进行序列测定和同源性分析；采用RT-PC方法观察其一在着床前胚胎的表达。结果：克隆发现18个基因和8个EST序列在GV卵中特异表达。RT-PCR显示该阳性克隆的基因在GV卵、MII卵、1-细胞胚胎和2-细胞胚胎有表达，其短片段是预期要扩增者，从MII卵开始表达下降；长片段在Gv卵、MII卵、1．细胞胚胎和2-细胞胚胎表达未见明显变化。长、短片段两端序列一致。结论：该基因是GV卵特异cDNA文库中的一个基因，且是母源性基因，提示该基因在卵母细胞成熟和合子基因激活中起一定作用。     

PeroxiredoxinII在小鼠卵巢中卵泡、GV卵、MII卵和植入前胚的表达和定位

目的　观察PeroxiredoxinII在小鼠卵巢中卵泡和植入前胚的表达与定位 ,为探讨PeroxiredoxinII在小鼠卵母细胞发育成熟和早期胚发育的作用提供依据。　方法　根据小鼠PeroxiredoxinIImRNA序列 (AccessionNo :BC0 0 2 0 34)设计引物 ,对小鼠生发泡完整卵细胞 (GV卵 )中的PeroxiredoxinII可能编码区进行扩增。同时 ,取生后 3d和 2月小鼠的卵巢 ,免疫细胞化学染色显示PeroxiredoxinII蛋白在卵泡中的分布。此外 ,运用RT PCR和免疫细胞化学染色方法观察PeroxiredoxinII在植入前胚的表达。　结果　从GV卵中扩增所得 6 84bp的cDNA序列 ,与小鼠PeroxiredoxinII具有 99%同源性 ;其推导的编码氨基酸序列与小鼠PeroxiredoxinII氨基酸序列完全相同。免疫细胞化学染色显示 :生后 3d小鼠卵巢内未见PeroxiredoxinII阳性反应 ;2月小鼠卵巢内阳性反应见于初级卵泡、次级卵泡和近成熟卵泡中卵母细胞的胞质。卵泡细胞未见PeroxiredoxinII阳性反应。RT PCR结果表明 :PeroxiredoxinIImRNA在GV卵中处高水平 ,从MII卵开始略有下降 ,在 4 细胞胚和早期囊胚期间未检到。免疫细胞化学染色显示 :GV卵、MII卵、1 细胞胚、2 细胞胚、4 细胞胚、8 细胞胚、桑椹胚和早期囊胚均呈PeroxiredoxinII阳性反应。　结论　PeroxiredoxinIImRN     

卵母细胞成熟障碍4例与同期卵母细胞大部分不成熟患者比较分析

目的分析4例体外受精-胚胎移植（IVF—ET）患者共8个辅助助孕周期,全部73个卵母细胞均处于GV、MetaphaseI期的可能原因。并与同期13例IVF—ET患者比较分析,比较组患者〉50％卵母细胞处于GV、MetaphaseI期。方法回顾分析本中心12年辅助助孕工作中出现的4例患者共8个周期所获卵母细胞均处于GV、MI的临床及实验室资料,并与同期13例超过50％卵母细胞处于GV、MI患者的临床、实验室资料进行比较。结果4例患者73个卵母细胞均处于GV或MI期,经体外成熟培养,24,48,72h仍无极体排出,停滞于GV、MI期。同期13例患者大部分卵母细胞不成熟,但体外培养后部分卵母细胞可进一步成熟,并可受精,获得妊娠。结论细胞和遗传机制引起卵母细胞成熟障碍,现有体外成熟培养方法尚无法促其成熟,目前赠卵是该类患者获得妊娠可供选择的助孕方法。但对于在控制性超促排卵中,出现大部分卵母细胞不成熟的患者可以通过延长促超排时间,增大hCG注射日卵泡直径,体外成熟培养等方法获得成熟的卵母细胞,获得妊娠。     

小鼠GV卵中母源基因的克隆和其中一个基因在植入前胚表达的研究

目的 克隆和筛选小鼠生发泡完整卵母细胞(GV卵)中母源基因，并检测其中一个基因在植入前不同发育阶段胚中的表达，初步了解该基因在卵母细胞发育成熟及早期胚发育中的作用。方法 应用抑制性消减杂交技术(SSH)，以生发泡完整的卵母细胞为检测子(tester)，8-细胞胚为驱赶子(driver)，建立GV卵中母源基因的cDNA文库。通过斑点杂交法进一步筛选GV卵中母源基因的cDNA文库，对阳性克隆进行序列测定和同源性分析；并采用RT-PCR方法观察其中1个阳性克隆的基因在着床前胚的表达。结果 克隆得到18个基因的cDNA序列和8个同源EST序列在GV卵中特异表达，包括PeroxiredoxinⅡ基因。RT-PCR显示PeroxiredoxinⅡ基因呈阶段性差异表达，在GV卵、MⅡ卵、1-胞胚和2-细胞胚有表达，但从MⅡ卵开始表达下降。而在4-细胞胚、8-细胞胚、桑椹胚和囊胚中不表达。结论 PeroxiredoxinⅡ基因是GV卵中母源基因cDNA文库中的一个基因，提示该基因在卵母细胞发育成熟和合子基因激活中起一定作用。     

p21活化激酶2在爪蟾卵母细胞的细胞质分裂过程中的作用

目的 研究p21活化激酶2(PAK2)在爪蟾卵母细胞的细胞质分裂中的作用.方法 以爪蟾卵母细胞为模型,利用特异性抑制PAK2活性的PAK2-N端(PAK2-NT)片段,显微注射爪蟾卵母细胞.荧光显微镜下比较PAK2-NT mRNA注射组和对照组卵母细胞生发泡破裂的发生.激光扫描共焦显微镜下,时间延迟摄影法观察两组卵母细胞的细胞质分裂过程中肌动蛋白和纺锤体的变化.结果 PAK2-NT mRNA注射组卵母细胞与对照组卵母细胞生发泡破裂发生相似,但PAK2-NT mRNA注射组卵母细胞未见细胞质分裂.结论 PAK2可能参与爪蟾卵母细胞的细胞质分裂过程.     

Renal dysplasia and asplenia in two sibs

A family is reported in which two sibs, one male and the other female, both died within 24 hours of birth with enlarged polycystic kidneys. Postmortem histology in the second child showed gross renal dysplasia. In both children the pancreas was enlarged, nodular and cystic but the liver appeared macroscopically normal. In the second child, histological examination confirmed pancreatic fibrosis with cystic dilation of ducts, but showed portal fibrosis with bile duct proliferation in the liver.
This combination of findings is very reminiscent of those in a girl and her brother reported by Ivemark et al. (1959). The children reported here also showed absence or hypoplasia of the spleen, cardiac anomalies and other features of the Ivemark syndrome (Ivemark 1955), a quite different, usually sporadic, congenital disorder. It is suggested that the children described here have a distinct lethal congenital disorder, probably inherited in an autosomal recessive manner.     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

What will studies of Fulani individuals naturally exposed to malaria teach us about protective immunity to malaria?

There are an estimated over 200 million yearly cases of malaria worldwide. Despite concerted international effort to combat the disease, it still causes approximately half a million deaths every year, the majority of which are young children with Plasmodium falciparum infection in sub-Saharan Africa. Successes are largely attributed to malaria prevention strategies, such as insecticide-treated mosquito nets and indoor spraying, as well as improved access to existing treatments. One important hurdle to new approaches for the treatment and prevention of malaria is our limited understanding of the biology of Plasmodium infection and its complex interaction with the immune system of its human host. Therefore, the elimination of malaria in Africa not only relies on existing tools to reduce malaria burden, but also requires fundamental research to develop innovative approaches. Here, we summarize our discoveries from investigations of ethnic groups of West Africa who have different susceptibility to malaria.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

HLA in North Colombia Chimila Amerindians

HLA-A,-B,-C,-DRB1 and -DQB1 alleles have been studied in Chimila Amerindians from Sabana de San Angel (North Colombian Coast) by using high resolution molecular typing. A frequent extended haplotype was found:HLA-A*24:02-B*51:10-C*15:02-BRB1*04:07-DQB1*03:02 (28.7%) which has also been described in Amerinndian Mayos Mexican population (Mexico, California Gulf, Pacific Ocean). Other haplotypes had already been found in Amerindians from Mexico (Pacific and Atlantic Coast), Peru (highlands and Amazon Basin), Bolivia and North USA. A geographic pattern according to HLA allele or haplotype frequencies is lacking in Amerindians, as already known. Also, five new extended haplotypes were found in Chimila Amerindians. Their HLA-A*24:02 high frequencies characteristic is shared with aboriginal populations of Taiwan; also, HLA-C*01:02 high frequencies are found in New Zealand Maoris, New Caledonians and Kimberly Aborigines from Australia. Finally, this study may show a model of evolutionary factors acting and rising one HLA allele frequency (-A*24:02), but not in others that belong to the same or different HLA loci.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.     

The universal muscular chamber. A basic unit of the genitourinary, cardiovascular, gastro-intestinal, skeletal, cutaneous, respiratory and other systems, endocameral hypertension; and spasm, the key to their idiopathic diseases and arthropathies

Starting with the integument, we see many organs are contractile sacs or multiples thereof, which tubes or bags constitute the major part of the entire body. Recognition of this basic unit and its characteristics sheds new light, individually and collectively, on many disorders previously considered unrelated. Muscular tears and perforations develop in the walls of these chambers, being no way peculiar to those organs, wherein, hydrochloric acid occurs. So, it is not necessary to explain the absence of excessive acid from patients who exhibit holes in the gastric, uterine, aortic, duodenal, rectal, pulmonary, retina, and other walls. Muscle, not acid is the great common factor relating idiopathic disorders in the gastrointestinal tract to each other and to similar diseases in other systems. When the units are linked together, the lesions tend to appear as arthropathies, i.e. at the joints. Rephrasing common-place observations, frees us from conventional, conceptual cul-de-sacs. An observation is only as good as its interpretation, so all possibilities must be considered, otherwise, we will remain blinded by our misconceptions.