融合蛋白 NrCAM-Fc的重组体的构建

目的：构建神经原相关的细胞粘附分子（NrCAM）和免疫球蛋白Fc片段融合基因的重组质粒并通过Baculovirus载体转染到昆虫细胞。方法：利用RT－PCR方法从神经母细胞瘤细胞系SK－N－SN获得NrCAM的信号肽区和跨膜区片段并直接克隆到pGEMT载体，经多次酶切、连接、转化、筛选获得NrCAM-Fc的重组副合基因，测序后将此基因通过Baculovirus载体转染至昆虫细胞。结果：多种酶切和测序结果表明NrCAM-Fc的序列与原序列符合率达98％以上，NrCAM与Fc连接处序列保证了Fc的读码框架（ORF）。结论：Baculovirus载体转染效率高，检测方法简便易行。     

MGA5 Health Utilities Index: Further Evidence of Responsiveness

Quality adjusted life years (QALYs) are well recognized as a valid measure for outcomes in cost-effectiveness analyses. However, it is difficult to obtain a summary utility score from health status measure such as the SF-36.
OBJECTIVE: To predict a summary utility score (represented by HUI) from the scores on the SF-36.
METHODS: A structural equation framework was applied to data collected from 1992 to 1995 on the Southern California Kaiser Permanente population (n = 5,794). An instrumental variable (IV) method mitigated the endogeneity in estimating the HUI(MarkII). Socioeconomic and disease variables were used as covariates. A split-sample analysis provided cross-validation.
RESULT: This model predicted 33.68% of the observed variance in HUI index scores with an adjusted R²of 0.3335. Observed HUI index scores were distributed with a mean of 0.7963 and std. deviation of 0.1796. Parameter estimates of most of the SF-36 components (except General Health & Social Functioning) showed statistical significance at α= 0.05 level. People with high chronic disease scores were found to have low SF-36 scores, and parameter estimates of this covariate were also found statistically significant at α= 0.05 level in all structural equations. However, all the socioeconomic variables showed statistical insignificance. Comparison of "Forecasting" and "Estimation" sub-samples showed satisfactory results during cross-validation.
CONCLUSION: Result of this study provides a quantitative link between two important measures of health status. The present model can be used to estimate overall health utility summary scores from previous studies using the SF-36.     

Evidence for the absorption of heparin by rat stomach.

Antithrombotic activity and heparin with endothelium are observed in rats when heparin is administered by the oral route. Peak endothelial concentrations at 6 min suggest rapid absorption. To identify the site of absorption, stomach and duodenum were isolated by tying the pyloric sphincter of male Wistar rats and heparin (unfractionated bovine lung, 60 mg/kg) was administered by stomach tube or injected into the duodenum. Heparin in plasma and aortic endothelium, collected within 15 min, was determined by densitometry following agarose gel electrophoresis with toluidine blue staining. Heparin was recovered in 5 of 10 endothelial (0.136+/-0.068 microg/cm(2)) and 6 of 10 plasma (0.06+/-0.02 microg/ml) samples when administered in the stomach and in 0 of 9 endothelial and 2 of 9 plasma (0.02+/-0.02 microg/ml) samples when injected into the duodenum. To further study heparin distribution, stomach layers were separated and analysed 15 min and 4h following heparin administration by stomach tube. Heparin was recovered in muscle and mucosal layers as well as washes indicating that heparin passes through stomach tissue. Heparin was also recovered from the portal vein, endothelium and lung. These results indicate that heparin is absorbed following oral administration and that the stomach is an important site of absorption.     

Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis

To determine whether reactive oxygen molecules could directly and reversibly increase the transfer of albumin across an endothelial barrier, we measured albumin transfer across monolayers of endothelium cultured on micropore filters before and after exposure to xanthine and xanthine oxidase. Xanthine and xanthine oxidase increased endothelial albumin transfer in a dose-dependent fashion. Parallel phase contrast and fluorescence microscopy demonstrated retraction of adjacent cells from one another and disruption of the actin filaments. The oxidant- induced increases in albumin transfer and changes in cell shape were reversed by removing xanthine oxidase and then incubating the monolayers for 3 1/2 hours in tissue culture media enriched with fetal bovine serum. However, incubation in tissue culture media without serum resulted in progressive injury and cell death. Hence, the brief exposure to oxidants initiated a progressive injury process that was reversed by incubation in serum. Because intracellular and extracellular calcium are important determinants of cell shape, and because some oxidized membrane lipids act as calcium ionophores, we asked whether oxidants altered endothelial calcium homeostasis. Xanthine-xanthine oxidase increased release of 45Ca++ from preloaded cells. The calcium antagonist lanthanum chloride prevented xanthine- xanthine oxidase increases in endothelial albumin transfer and prevented the changes in cell shape; chelation of extracellular calcium inhibited lysis of endothelium by xanthine-xanthine oxidase; and the calcium ionophore A23187 increased endothelial albumin transfer and mimicked the oxidant-induced changes in cell shape. Lanthanum chloride inhibited these effects of A23187. These data suggest that oxygen radicals can reversibly increase endothelial permeability to macromolecules, that this is associated with reversible changes in endothelial cell shape and actin filaments, and that the changes in cell shape are related to oxidant-induced changes in endothelial calcium homeostasis.     

Three patients with structurally abnormal X chromosomes, each with Xq13 breakpoints and a history of idiopathic acquired sideroblastic anemia

Structural abnormalities of the X chromosome are rarely found in neoplastic disorders. We describe three patients with a history of idiopathic acquired sideroblastic anemia (IASA); each one had an abnormal clone of cells in the bone marrow, characterized by a structurally abnormal X chromosome. In two of these patients, the predominant karyotype was 47,X,2idic(X)(q13); in the other patient, it was 46,X,t(X;11)(q13;p15). Inasmuch as all three of these cases involved chromosome band Xq13, as did two previously published cases, we suggest that band Xq13 may be more prone to structural rearrangement than other X chromosome bands in hematologic disorders. The common Xq13 chromosome breakpoint and clinical presentation (IASA) among these three patients and the occurrence of an X-linked type of sideroblastic anemia may suggest that an association exists between X chromosome abnormalities and IASA. Perhaps alteration of a gene or chromosome structure in or near band Xq13 predisposes to development of IASA. The fact that two of these patients had preleukemia and the third had overt acute leukemia may imply that patients with IASA and X chromosome abnormalities have a poor prognosis. Cases of IASA without associated X chromosome abnormalities are known; thus, if an association between IASA and an abnormal X chromosome does exist, most likely it involves only some patients with IASA.     

Correlation between patterns of horizontal connectivity and the extend of short-term representational plasticity in rat motor cortex

Plasticity of representational maps in adult cerebral cortex has been documented in both sensory and motor cortex, but the anatomical basis for cortical plasticity remains poorly understood. To investigate horizontal connectivity in primary motor cortex (M1) as a putative anatomical substrate for short-term, functional plasticity of adult motor cortical representations, a combination of electrical stimulation and biocytin labeling was used to examine pre-existing patterns of intrinsic connections in adult rat M1 in relationship to the pattern of reorganization of the motor movement may induced by transection of the contralateral facial nerve. Two hours after nerve cut, small, circumscribed regions of the forelimb representation expanded medially into territory previously devoted to the vibrissae representation. Outside of this novel, expanded forelimb region, no forelimb movement could be evoked from the former vibrissae representation at any time over the period of hours tested, thus representing silent cortex. Injections placed into vibrissae cortex representing the newly expanded forelimb representation gave rise to labeled axons and dense terminal fiber labeling which crossed the forelimb/vibrissae border and extended up to 1.2 mm within the low-threshold forelimb representation. In contrast, injections placed into silent vibrissae cortex gave rise to labeled axons and terminal boutons which remained mostly restricted to the original vibrissae representation, with only sparse projections that crossed into the low-threshold forelimb representation. Thus, these results suggest that the extent of short-term, functional reorganization of M1 induced within the first several hours following peripheral nerve cut is mediated, and constrained, by an anatomical framework of pre-existing, horizontal projections which traverse representation borders.      

Evidence from endothelium of gastric absorption of heparin and of dextran sulfates 8000

Heparin, hydrogenated dextran sulfate 8000 (Usherdex 8), and dextran sulfate 8000 were administered to rats, and the total drug was separated and determined in endothelium and plasma. A large amount of each drug was recovered from endothelium 2.4 and 6 minutes after intravenous injection. This accounted for the drug missing from plasma. The drugs in water were placed in the stomach by catheter. All three drugs were recovered from the endothelium and identified unchanged by electrophoresis and specific staining. The amounts that were recovered at 2.4 and 6 minutes were equivalent to most of the drug administered. Thus heparin, Usherdex 8, and dextran sulfate 8000 enter the body immediately on oral administration. At longer time intervals after intravenous and oral administration, much of each drug was not demonstrable in endothelium by the method used. Some drug could be detected in endothelium after 4 hours. After oral administration, plasma levels of each drug were rarely more than 0.5% of the dose. Formalin-alcohol was applied to the jugular veins of anesthetized rats to produce a thrombus, (see Blake et al. J Clin Path 1959;12:118-22) and the drugs were immediately introduced into the stomach. Four hours later the injured veins were inspected for thrombi. Incidence of thrombotic plug was 80% in rats that received saline solution, 4% with Usherdex 8, 0% with dextran sulfate 8000, and 0% with heparin. Usherdex 8, dextran sulfate 8000, and heparin demonstrate low, moderate, and high in vitro anticoagulant activity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)