直接扩增和鉴别粪标本中致病性与非致病性溶组织内阿米巴的DNA

取定量的HK-9株溶组织内阿米巴滋养体放入受检者的0.1g粪便中,再加DTT、KOH等在65℃孵育15min,后用HCl中和,并以酶/氯仿/异戊醇振荡抽提DNA。同时再取受检者粪便0.1g以NaCl洗涤,离心,蛋白酶K消化,抽提其DNA。用以rRNA为模板设计的致病性与非致病性引物进行双向PCR,PCR产物以含0.2μg/ml溴乙锭的1.3％的琼脂糖凝胶电泳显示,并用限制性内切酶DraⅠ和Sau 96Ⅰ进行消化。     

单克隆抗体对溶组织内阿米巴吞噬红细胞的影响

用具有抗细胞膜活性和抗细胞核、胞浆活性的单克隆抗体３Ｆ７和４Ｇ６与致病性溶组织内阿米巴ＨＭ－１∶ＩＭＳＳ株滋养体３７℃孵育，在孵育即刻、５ｍｉｎ、１０ｍｉｎ加入红细胞，检测滋养体吞噬红细胞的能力，并以正常小鼠血清和抗克氏锥虫抗体ＴＣＥ０４作为对照。结果，经单克隆抗体３Ｆ７孵育即刻的ＨＭ－１∶ＩＭＳＳ虫株滋养体吞噬红细胞能力明显减弱（Ｐ＜０．００１），而４Ｇ６抑制滋养体吞噬红细胞的作用不如前者明显。提示，抗细胞膜单克隆抗体对抑制滋养体吞噬红细胞有显著作用，但随着时间的推移，抑制作用明显减弱。     

Activity of synthetic enzymes of tetrahydrobiopterin in the human placenta

Tetrahydrobiopterin (BH4) is an essential co-factor for nitric oxide (NO) synthase (NOS) and regulates the production of NO, or endothelium-derived relaxation factor. Although NOS is highly expressed in the placenta and NO plays a critical role in the regulation of feto-placental circulation, the mechanism maintaining the level of BH4 is not known. To investigate the de novo synthesis of BH4 in the human placenta, the activity of guanosine triphosphate cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR) in the chorionic tissue during the first, second and third trimester of pregnancy was analyzed. GTPCH activity was the lowest of the three enzymes and became negligible after the second trimester. There was no significant change in PTPS activity throughout pregnancy. Although SR activity decreased significantly after the second trimester, the levels remained abundant throughout pregnancy. These results showed that GTPCH is a rate-limiting enzyme and the total activity of the de novo synthesis of BH4 is negligible in the mature placenta after the second trimester when fetal growth is accelerated. The present study suggests that the level of BH4 in the placenta depends principally on the system other than de novo synthesis. The salvage pathway is considered the most potent system, which is formed by the transfer of the substrates from the fetus and their enzymatic conversion to BH4 in the placenta.     

Receptors and transporter for serotonin in Merkel cell-nerve endings in the rat sinus hair follicle. An immunohistochemical study

Serotonin (5-HT) has been a candidate for neurotransmitters in cutaneous type I mechanoreceptors (i.e., Merkel cell-nerve endings). Although recent electrophysiological studies have suggested the presence of the 5-HT2 and 3 receptors in the Merkel cell-nerve endings, the histological localization of these receptors are obscure. We thus immunohistochemically examined the presence of 5-HT1, 2, 3 receptors in Merkel cell-nerve endings in sinus hair follicles of the rat whisker pad. We also studied the immunohistochemical localization of the 5-HT transporter to confirm the site of 5-HT secretion. For this purpose, we used antibodies for the 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C and 5-HT3 receptors, and for the 5-HT transporter, as well as antibodies for cytokeratin 20 (as a marker of Merkel cells) and neurofilament H (a marker of type I sensory nerve terminals). The immuno-stained sections were analyzed under a laser-scanning microscope. It was found that the sensory nerve terminals in the Merkel cell-nerve endings showed strong positive immunoreactions of 5-HT1A and 1B receptors but not 5-HT2A, 2C, and 3 receptors. Furthermore, both the Merkel cells and related axon terminals showed strong immunoreactions of the 5-HT transporter. These findings support the idea that 5-HT molecules are released from the Merkel cells during mechanical reception and indirectly regulate neural actions of sensory neurons via 5-HT1 receptors. The localization of the 5-HT transporter found in this study also suggests a possibility that axon terminals in the Merkel cell-nerve endings also release 5-HT.     

Genomic alterations in primary cutaneous melanomas detected by metaphase comparative genomic hybridization with laser capture or manual microdissection: 6p gains may predict poor outcome

To clarify the correlation of genomic alterations with clinical and histological features, we performed metaphase comparative genomic hybridization analysis on 20 primary cutaneous melanomas, which were obtained by laser capture or manual microdissection, and 16 melanoma cell lines. There were no differences in the average number of aberrations between acral melanomas (AM) and non-AM, although gains of 5q and 11q13 were more frequent (P=0.05) and 10q loss was less frequent (P=0.01) in AM than in non-AM. Although tumor thickness is considered a measurable estimate of clinical expression, there were no differences in the average number of aberrations among 4 groups, classified by thickness of the tumor. While the majority of aberrations were equally distributed among the 4 groups, 6p gains were found only in the thickest tumors. Patients with 6p or 1q gains had a lower overall survival rate than those without them (P=0.0002 or P=0.013). While gains of 1q, 2q, 3p, 3q, 7q, 20p, and 20q were more frequent in the cell lines than in the primary tumors (P<0.01), losses of 6q, 9p, 10p, and 10q were equally found in both cell lines and primary tumors. The present study showed that chromosomal aberrations had already occurred in the thinner tumors, and that 6p and 1q gains may be a prognostic factor.     

三种蚊唾腺蛋白IgE亲和成分的初步分析

从我国分布较广的三种蚊中华按蚊、淡色库蚊和白纹伊蚊中分离出唾液腺 ,制成唾腺蛋白粗提物 ,再应用ELISA、竞争抑制ELISA和免疫印迹试验分析其与IgE结合的组分。结果显示三种蚊唾腺蛋白含能与IgE结合的成分 ,以白纹伊蚊反应所呈现的A值最高 ;免疫印迹在三种蚊均显示 4条阳性带 ,中华按蚊和淡色库蚊相似 ,白纹伊蚊有 3条带差异 ,但其相对分子质量为 30 0 0 0～ 310 0 0组分阳性条带均出现于三种蚊唾腺蛋白     

Role of ionotropic glutamatergic and GABAergic inputs on the firing activity of neurons in the external pallidum in awake monkeys

The neurons in the external segment of the pallidum (GPe) in awake animals maintain a high level of firing activity. The level and pattern of the activity change with the development of basal ganglia disorders including parkinsonism and hemiballism. The GPe projects to most of the nuclei in the basal ganglia. Thus exploring the mechanisms controlling the firing activity is essential for understanding basal ganglia function in normal and pathological conditions. To explore the role of ionotropic glutamatergic and GABAergic inputs to the GPe, unit recordings combined with local injections of receptor antagonists were performed in awake monkeys. Observations on the effects of local application of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate antagonist 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide, the N-methyl-D-aspartic acid (NMDA) antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, and the GABAA antagonist gabazine as well as the effects of muscimol blockade of the subthalamic nucleus on the spontaneous firing rate, firing patterns, and cortical stimulation induced responses in the GPe suggested the following: sustained glutamatergic and GABAergic inputs control the level of the spontaneous firing of GPe neurons; both AMPA/kainate and NMDA receptors are activated by glutamatergic inputs; some GPe neurons receive glutamatergic inputs originating from areas other than the subthalamic nucleus; no GPe neurons became silent after a combined application of glutamate and GABA antagonists, suggesting that GPe neurons have intrinsic properties or nonionotropic glutamatergic tonic inputs that sustain a fast oscillatory firing or a combination of a fast and a slow oscillatory firing in GPe neurons.     

Expression of cytoplasmic TFF2 is a marker of tumor metastasis and negative prognostic factor in gastric cancer

Trefoil factor family 2 (TFF2) is a small peptide constitutively expressed in the gastric mucosa, where it plays a protective role in restitution of gastric mucosa. TFF2 has also been shown to be expressed in some gastric cancers, but its role in tumor metastasis and patient prognosis has not been examined. In this study, we examined TFF2 expression at both the mRNA and protein levels and correlated these results with the clinicopathologic characteristics and prognosis of gastric cancer patients. Among the 144 curatively resected samples, 43 (30%) were positive for TFF2. TFF2 expression was preferentially observed in the infiltrating tumor cells sparing the superficial cells. Significantly increased expression of TFF2 was noted in large tumors of the diffuse type. An increased prevalence of TFF2 expression was also found in tumors with advanced T and N stage and in patients with lymphatic and venous invasion. Accordingly, patients with TFF2-expressing tumors had a significantly worse disease-free survival, and in multivariate analysis, this finding remained significant as an independent prognostic factor. Taken together, our results suggest that TFF2 expression may play a role in gastric cancer invasion and as such could be a useful target for therapeutic intervention.     

伴有肌样/肌纤维母细胞性分化的隆突性皮纤维肉瘤的临床病理分析

目的 探讨隆突性皮纤维肉瘤（ＤＦＳＰ）中肌样／肌纤维母细胞性分化的本质及其临床病理学意义。方法 采用常规ＨＥ切片对１２４例ＤＦＳＰ进行筛选,对6例伴有肌样／肌纤维母细胞性分化的ＤＦＳＰ病例进行免疫组织化学标记,其中２例加做电镜检测。结果 肌样／肌纤维母细胞性分化多出现在纤维肉瘤型ＤＦＳＰ（ＦＳ－ＤＦＳＰ）中,表现为肿瘤周边部或肿瘤内散在性分布的深嗜伊红色小结节或短要束,由梭形细胞组成,细胞多无异型性,核分裂象也罕见,形态上似平滑肌细胞或肌纤维母细胞。免疫组织化学标记显示肌样区域细胞表达α－平滑肌肌动蛋白和肌物质特异性肌动抗原,不表达ＣＤ３４;电镜观察证实细胞含有质膜下微丝束、局灶性致密体及微胸饮囊泡样结构,与肌纤维母细胞相一致,结论 ＤＦＳＰ中的肌样／肌纤维母细胞性分化可能是间质中肌纤维母细胞增生的结果,并非代表了瘤细胞的真性肌纤维母细胞性分化。     

Improved affinity of a human anti-Entamoeba histolytica Gal/GalNAc lectin Fab fragment by a single amino acid modification of the light chain

We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose- and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser91 and Arg96 in the third complementarity-determining region of the light chain with other amino acids. The screening of 200 clones of each exchange by an indirect fluorescent antibody test showed that 14 clones for Ser91 and nine clones for Arg96 reacted strongly with E. histolytica trophozoites. Sequence analyses revealed that the substituted amino acids at Ser91 were Ala in five clones, Gly in three clones, Pro in two clones, and Val in two clones, while the amino acid at position 96 was substituted with Leu in three clones. The remaining eight clones exhibited no amino acid change at position 91 or 96. These mutant Fab fragments were purified and subjected to a surface plasmon resonance assay to measure the affinity of these proteins to the cysteine-rich domain of lectin. Pro or Gly substitution for Ser91 caused an increased affinity of the Fab, but substitution with Ala or Val did not. The replacement of Arg96 with Leu did not affect affinity. These results demonstrate that modification of antibody genes by recombination PCR is a useful method for affinity maturation and that amino acid substitution at position 91 yields Fabs with increased affinity for the lectin.