LuSIV cells: a reporter cell line for the detection and quantitation of a single cycle of HIV and SIV replication

A single cycle of viral replication is the time required for a virus to enter the host cell, replicate its genome, and produce infectious progeny virions. The primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), require on average 24 h to complete one cycle of replication. We have now developed and characterized a reporter assay system in CEMx174 cells for the quantitative measurement of HIV/SIV infection within a single replication cycle. The SIV(mac)239 LTR (-225 --> +149) was cloned upstream of the firefly luciferase reporter gene and this reporter plasmid is maintained in CEMx174 cells under stable selection. This cell line, designated LuSIV, is highly sensitive to infection by primary and laboratory strains of HIV/SIV, resulting in Tat-mediated expression of luciferase, which correlates with viral infectivity. Furthermore, manipulation of LuSIV cells for the detection of luciferase activity is easy to perform and requires a minimal amount of time as compared to current HIV/SIV detection systems. The LuSIV system is a powerful tool for the analysis of HIV/SIV infection that provides a unique assay system that can detect virus replication prior to 24 h and does not require virus to spread from cell to cell. Thus these cells can be used for the study of replication-deficient viruses and the high throughput screening of antivirals, or other inhibitors of infection.     

Identification and comparison of residues critical for cell-adhesion activities of two neutrophil CD66 antigens, CEACAM6 and CEACAM8

CEACAM6 (CD66c) and CEACAM8 (CD66b) are cell-adhesion proteins on neutrophils that belong to the human carcinoembryonic antigen (CEA) family. CEACAM6 reveals homophilic adhesion and heterophilic adhesion to other CEACAM family antigens including CEACAM8, CEACAM1, and CEA, whereas CEACAM8 exhibits only heterophilic adhesion to CEACAM6. Here, we investigated and compared structural requirements for the homophilic adhesion of CEACAM6 and heterophilic adhesion between CEACAM6 and CEACAM8 at the amino acid level by using CHO transfectants expressing their mutant and chimeric proteins. The NH(2)-terminal domain (N-domain) of CEACAM6 expressed on a CHO cell was suggested to bind the N-domain of CEACAM6 or CEACAM8 on the opposing cell. By homologue-scanning mutagenesis, we found that the locations of the sequences critical for the adhesion of CEACAM6 to itself and to CEACAM8 are overlapped and that they are highly similar but not identical to the locations of the residues previously shown to be essential for the binding of CEACAM antigens to Opa proteins of pathogenic NEISSERIAE: Our findings imply that subtle differences in the N-domain sequences determine the specificity of the CEACAM antigens on neutrophils for interaction with the same or different CEACAM antigens and the bacterial proteins.     

肺淋巴管平滑肌瘤病临床病理学观察

目的 探讨肺淋巴管平滑肌瘤病临床、病理特征。方法 对5例肺淋巴管平滑肌瘤病临床资料进行收集分析,HE切片观察,采用免疫组织化学(SP法)检测平滑肌肌动蛋白(SMA)、HMB45、基质金属蛋白酶(MMP)2、孕激素受体(PR)、雌激素受体(ER),并进行文献复习。结果 肺淋巴管平滑肌瘤病是原因不明的肺部疾病,只发生在女性,特别是绝经前妇女。临床表现为呼吸困难,咯血,气胸和乳糜胸等。病理学检查显示不同成熟度平滑肌细胞在细支气管壁、肺泡壁、淋巴管壁和血管壁周围增生,肺实质呈囊性变。增生的平滑肌细胞免疫组织化学5例SMA、HMB45、MMP2均阳性;1例的ER和PR均阳性,1例仅ER阳性,1例仅PR阳性,1例的ER和PR均阴性。结论 育龄期妇女如反复出现自发性气胸、咯血、活动后呼吸困难应考虑肺淋巴管平滑肌瘤病的可能,病理检查可确定肺淋巴管平滑肌瘤病的诊断。     

带臂外侧上皮神经及其营养血管筋膜皮瓣的应用解剖

目的:为带臂外侧上皮神经及其营养血管筋膜皮瓣提供解剖学基础.方法:32例经灌注红色乳胶的成人上肢标本,对臂外侧上皮神经及其营养血管等进行了较详细的应用解剖学研究.结果:臂外侧上皮神经在均由腑神经发出,起点横径为1.5±0.4mm,在三角肌深方斜向外下3.6±1.1cm从该肌后缘中1/3浅出肌间隔,分为上支和下支,分布于三角肌后部、外侧部和臂外侧上部.该神经的营养血管起源于旋肱后动脉,起点外径为0.9±0.4mm;其行程、分支和分布均同在神经,供血范因为14.8×9.8cm~2,并与周围的皮动脉存在丰富吻合.结论:带臂外侧上皮神经及其营养血管筋膜皮 瓣可视受区需要设计成游离瓣或旋转瓣,用于修复邻近部位、手或颌面部缺损.     

Comparison of immunohistochemical markers in the differential diagnosis of adrenocortical tumors: immunohistochemical analysis of adrenocortical tumors.

Most adrenocortical tumors (ACTs) can be diagnosed directly by a combination of morphologic features and clinical findings. However, sometimes it may be difficult to distinguish ACTs from other neoplasms such as pheochromocytomas and some metastatic tumors, particularly for small biopsy specimens because they may be morphologically similar. Expression of calretinin has recently been suggested as a valuable immunomarker for the differential diagnosis between ACTs and other tumors; however, its diagnostic value is still under debate. To determine the diagnostic value of calretinin in Chinese patients with adrenocortical and non-ACTs, we employed both polyclonal and monoclonal anticalretinin to characterize the expression of calretinin in adrenal tissues and compared its expression with that of inhibin alpha, Melan-A, cytokeratin, or CD99 by immunohistochemistry in tissue microarrays and standard tissue sections of 414 specimens. Our results revealed that calretinin was expressed by adrenocortical cells, but not by the other cells tested and the percentage of calretinin-positive ACTs reached 99% when stained with polyclonal antibodies, which was higher than that with monoclonal anticalretinin (91.3%), anti-Melan-A (90.3%), antiinhibin alpha (81.6%). In addition, our results also revealed that ACTs were stained by cytokeratin (AE1/AE3) with variable degrees (58.7%). Furthermore, unlike anti-Melan-A that stained all metastatic malignant melanoma, anticalretinin did not recognize other tested tumors. Therefore, immunohistologic staining with polyclonal anticalretinin is more sensitive than other antibodies tested for the diagnosis of ACTs. However, monoclonal anticalretinin appeared to be more specific. Importantly, our data suggested that the fried-egg-like staining pattern, but not the mere cytoplasmic staining, was characteristic of anticalretinin staining in adrenocortical tissues. Notably, a few anticalretinin negative-ACTs were stained by other immunomarkers that we tested. Thus, the combinational characterization of calretinin (either by polyclonal or monoclonal antibody), inhibin alpha, and Melan-A expression is of great significance in the differential diagnosis of ACTs.     

新鲜组织块标本经乙醇直接固定二年的流式细胞术DNA分析

本文报道了一种实体组织用于DesoxyribonucleicAcid(DNA)分析的Flowcy tometry(FCM) )样品保存新方法 ,即新鲜组织块乙醇直接固定法。所用样品为头颈部肿瘤手术切除的新鲜标本 3 0例 ,每例标本重约 0 5～ 1 g ,均等分为两份 ,1份置 70 %乙醇直接固定 ,室温放置 ,待两年后FCM检测。另一份置生理盐水中立即行流式细胞术DNA分析。两组样品经机械法制成单细胞悬液 ,PI染色后上机检测。结果显示 :从两组样品的DNA直方图分析 ,CV(coefficiencyofvariation)值、GO/G1、S及G2 /M各期比率无显著差异 (P >0 0 5)。我们认为在样品收集短期内难以完成 ,需积累保存 ,或需保存半年以上者 ,且不具备低温冷冻设备的条件下 ,新鲜组织块乙醇直接固定法是优于将组织块先制备成单细胞悬液再行乙醇固定的流式细胞术DNA样品保存方法     

可切削渗透陶瓷玻璃料对不同堆积密度氧化铝基体的渗透

探讨了可切削渗透陶瓷 (Infiltration of Machinable- infiltrated- ceramic,MIC)渗透玻璃在不同堆积密度氧化铝基体中的渗透情况及渗透后复合体的颜色表达。在 110 0℃保温 2 h,将玻璃渗透到不同堆积密度的氧化铝基体中 ,分别测得其渗透深度和颜色参数。扫描电镜观察玻璃—氧化铝复合体断面。玻璃渗透深度的平方与氧化铝堆积密度有直线负相关关系 ,最小渗透深度为 3.0 92 m m。复合体的颜色系数与氧化铝堆积密度无相关性。渗透复合体在断裂过程中可见 :裂纹偏转、晶体拔出和穿晶断裂。本实验证实 MIC渗透玻璃的渗透性能达到临床要求。渗透后的复合体颜色稳定 ,强度可靠     

Adhesion contact dynamics of HepG2 cells on galactose-immobilized substrates

The specific recognition between asialoglycoprotein receptor and galactose ligand at cell-substrate interfaces has been shown to mediate hepatocyte adhesion and maintain liver specific functions of hepatocytes. Conventionally, the success of hepatocyte attachment on engineered tissue scaffold is inferred from the degree of two-dimensional cell spreading that is measured by transmitted light microscopy. However, the actual contact mechanics and adhesion strength of hepatocytes during two-dimensional cell spreading has not been elucidated due to lack of biophysical probe. In this study, a novel biophysical technique known as confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy is utilized to probe the adhesion dynamics, contact mechanics and two-dimensional spreading kinetics of HepG2 cells on galactose immobilized and collagen gel coated substrates. C-RICM demonstrates that HepG2 cells form strong adhesion contacts with both galactose-immobilized surfaces and collagen gel coated substrates. Moreover, HepG2 cells maintain their compact shapes in the presence of asialoglycoprotein receptor-mediated recognition while they become exceedingly spread under integrin-mediated adhesion on collagen gel coated substrate. The initial rate of adhesion contact formation and the steady-state adhesion energy of HepG2 cell population are highest on substrate conjugated with galactose ligand via a longer spacer. The adhesion dynamics and final adhesion energy of HepG2 cells depends both on the type of ligand-receptor interaction and the length of spacer between the ligand and substrate. Most importantly, new biophysical insights into the initial hepatocyte attachment that are critical for hepatocyte culture are provided through the decomposition of two-dimensional spreading and adhesion contact formation on bio-functional substrates.     

Molecular cloning and sequence analysis of full-length cDNAs encoding new group of Cyn d 1 isoallergens

BACKGROUND: Cyn d 1, the major allergen of Bermuda grass pollen, contains some acidic/basic isoforms. The N-terminal amino acid sequences of some acidic Cyn d 1 isoforms were found to be different from those of Cyn d 1 cDNA clones identified previously. METHODS: A predicted 17-meric oligonucleotide probe was designed to fish the unidentified isoallergen cDNAs out of BGP cDNA library. The reactive clones were isolated and verified by sequencing. Two of them were expressed in the yeast Pichia pastoris to obtain recombinant Cyn d 1 proteins. RESULTS: All four cDNA clones encode the full-length Cyn d 1 with mature proteins of 244 amino acid residues. A 97-99% identity was found among the deduced amino acids of these four clones while an 86% identity was elicited between the four clones and the ones previously identified. The predicted isoelectric focusing (pI) values of the newly identified Cyn d 1s are acidic while pIs of the previously identified Cyn d 1s are basic. The two recombinant acidic Cyn d 1 proteins possess the epitopes recognized by mouse and rabbit polyclonal anti-Cyn d 1 antibodies, and have human IgE-binding capacity as revealed by immunodot assay. CONCLUSIONS: The present study identified full-length cDNAs encoding new isoallergens of Cyn d 1, and separated Cyn d 1 gene into an acidic group and a basic group.