On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual''s level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.¹ These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,² lipid-^{3, 4} and RAGE-induced⁵ inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-α. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.     

Inhibitory effect of cortisone acetate on the stimulation of rat liver cytosol l-serine Pyruvate aminotransferase by dibutyryl adenosine 3′,5′-monophosphate

An injection of cortisone acetate at a dose of 5 mg/100 g body weight concomitant with dibutryl cyclic AMP prevents the increase in the activity of rat liver cytosol serine aminotransferase (L-serine: pyruvate aminotransferase, EC 2.6.1.51) elicited by the nucleotide with a lag of about 2 h. If the glucocorticoid is given 2 h prior to the nucleotide inducer, the lag disappears. The inhibitory effect of cortisone acetate gradually decays and is no longer detectable 12 h following its administration. Theophylline, insulin and glucose at doses which affect significantly the level of tyrosine aminotransferase, have no effect on the level of serine aminotransferase and on the cortisone inhibition. The inhibitory effect of the glucocorticoid on the dibutyryl cyclic AMP-mediated increase in serine aminotransferase diminishes with the age of animals. Increase in the enzyme activity by a single dose of glucagon can also be inhibited by cortisone acetate and actinomycin D as in the case with dibutyrl cyclic AMP as an inducer. The possibility of the existence of a specific inhibitory factor which is formed in response to cortisone acetate is discussed.     

65Zinc uptake from blood into brain and other tissues in the rat

Zinc is essential for normal growth, development and brain function although little is known about brain zinc homeostasis. Therefore, in this investigation we have studied⁶⁵Zn uptake from blood into brain and other tissues and have measured the blood-brain barrier permeability to⁶⁵Zn in the anaesthetized rat in vivo. Adult male Wistar within the weight range 500–600 g were used.⁶⁵ZnCl₂ and [¹²⁵I]albumin, the latter serving as a vascular marker, were injected in a bolus of normal saline I.V. Sequential arterial blood samples were taken during experiments that lasted between 5 min and 5 hr. At termination, samples from the liver, spleen, pancreas, lung, heart, muscle, kidney, bone, testis, ileum, blood cells, csf, and whole brain were taken and analysed for radio-isotope activity. Data have been analysed by Graphical Analysis which suggests⁶⁵Zn uptake from blood by all tissues sampled was unidirectional during this experimental period except brain, where at circulation times<30 min,⁶⁵Zn fluxes were bidirectional. In addition to the blood space, the brain appears to contain a rapidly exchanging compartment(s) for⁶⁵Zn of about 4 ml/100g which is not csf.     

Blood-brain exchange routes and distribution of65Zn in rat brain

Zinc is essential for normal development and function of the CNS although much is to be learned about brain Zn homeostasis. In these experiments adult male Wistar rats within the weight range 500–600 g were used. Ventriculo-cisternal perfusion was performed to allow the measurement of⁶⁵Zn fluxes between blood and csf across the choroid plexuses. Blood-brain or blood-cerebrospinal fluid barrier permeability to⁶⁵Zn has been determined by graphical analysis in experiments that lasted between 5 and 180 minutes. Cerebral capillary permeability to⁶⁵Zn was found to be low with a K_in of about 5×10^–4ml/min/g. Choroid plexus permeability to⁶⁵Zn was about 12 fold greater, although Zn influx to brain via this route was <5% that across cerebral capillaries. The autoradiographic distribution of⁶⁵Zn in brain showed regional variation with lowest levels in white matter and high levels in the dentate gyrus and hippocampus.     

Dissimilatory hexaheme c nitrite reductase of “Spirillum” strain 5175: purification and properties

When grown with nitrate as terminal electron acceptor both the soluble (periplasm, cytoplasm) and the membrane fraction of Spirillum strain 5175 exhibited high nitrite reductase activity. The nitrite reductase obtained from the soluble fraction was purified 76-fold to electrophoretical homogeneity. The enzyme reduced nitrite to ammonia with a specific activity of 723 mol NO _inf2 ^sup- × (mg protein × min)^-1. The molecular mass was 58±1 kDa by SDS-PAGE compared to 59±2 kDa determined by size exclusion chromatography under nondenaturing conditions. The enzyme (as isolated) contained 5.97±0.15 heme c molecules/M_r 58 kDa. The absorption spectrum was typical for c-type cytochrome with maxima at 280, 408, 532 and 610 nm (oxidized) and at 420, 523 and 553 nm (dithionite-reduced). The enzyme (as isolated) exhibited a complex set of high-spin and lowspin ferric heme resonances with g-values at 9.82, 3,85, 3.31, 2.95, 2.30 and 1.49 in agreement with data reported for electron paramagnetic resonance spectra of nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli.Abbreviations DNRA dissimilatory nitrate reduction to ammonia - EPR electron paramagnetic resonance - PAGE polyacrylamide gel electrophoresis - NaP_i sodium phosphate - SDS sodium dodecylsulfate     

Status of reduced glutathione in primary cultures of rat hepatocytes and the effect on conjugation of benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide

The intracellular level of reduced glutathione (GSH) and GSH conjugation have been investigated in primary cell cultures of hepatocytes isolated from control rats, phenobarbitone (PB) and 3-methylcholanthrene (MC) treated rats. The data demonstrate that in all cell cultures the GSH concentrations show a triphasic pattern: (i) within 1 h of culture an initial marked decrease to 50% of the levels found in fresh hepatocytes; (ii) recovery of GSH concentrations to above the levels observed in fresh cells. This occurs after 6 h in culture with control cells and after 10-24 h with cells from either PB or MC treated rats and was most prominent in cells from PB-treated rats. (iii) A slow decline to between 30 and 40 nmol GSH/mg protein from 24 to 96 h in culture. Synthesis of GSH was slower in cultured cells from PB treated rats and this was confirmed by the resynthesis rates when diethylmaleate (DEM) was used to deplete GSH. The formation of GSH conjugates with racemic 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was measured in control cells in suspension and after 3 and 24 h in culture. Despite the decrease in GSH concentrations observed between 1 and 4 h after culture, the conjugation rates were not decreased.     

Differential Expression of a Mycobacterium tuberculosis Protein Recognized by a Mouse Monoclonal Antibody

Murine monoclonal antibodies were produced against Mycobacterium tuberculosis (Mtb) using standard hybridoma procedures. By a whole cell enzyme-linked immunosorbent assay (ELISA), one monoclonal antibody (mAb), HB28, demonstrated high level specific reactivity to Mtb. Western blot analysis demonstrated reactivity to a single 65 kDa Mtb protein in the cell wall extract and culture filtrate. HB28 mAb appears to be recognizing a 65 kDa Mtb protein that is over-expressed by Mtb but not other species under certain culture conditions. Differential expression and detection of this protein by HB28 mAb may have potential for diagnostic applications.     

生物素标记HBV RNA探针的制备及应用

本文首次采用ＳＰ６５特殊质粒与人类的乙型肝炎病毒ＤＮＡ重组,制备了Ｂｉｏ－ＨＢＶ ＲＮＡ探针,能特异地与ＨＢＶ ＤＮＡ杂交,将该探针与缺口转移方法标记的Ｂｉｏ－ＨＢＶ ＤＮＡ探针进行了比较,结果显示出Ｂｉｏ－ＨＢＶ ＲＮＡ探针比Ｂｉｏ－ＨＢＶ ＤＮＡ探针的敏感性提高１０倍,并分别应用两种探针同时检测７０例乙肝病人血清中ＨＢＶ ＤＮＡ,阳性率各为３１．４２％、２８．５７％（Ｐ＞０．２５）。对Ｂｉｏ－     

Neuronotypic Differentiation Results in Reduced Levels and Altered Distribution of Synaptophysin in PC 12 Cells

Abstract: Several synaptic vesicle proteins including synap-tophysin and p65/synaptotagmin are expressed by the pheochromocytoma cell line PC12. Stimulation of these cells with nerve growth factor for 7 days induces morphologic neuronotypic differentiation, but the levels of synaptophysin are markedly reduced. Stimulation with cyclic AMP analogs also produces neuronotypic differentiation of PC12 cells, and the degree of morphologic differentiation induced by these agents parallels their ability to effect reduction in synaptophysin levels. By contrast, levels of p65/synaptotagmin are increased following neuronotypic differentiation. The contrasting effects of neuronotypic differentiation on levels of synaptophysin and p65/synaptotagmin indicate potential differences in the regulation of these proteins in PC12 cells. Immunocytochemical labeling of undifferentiated PC12 cells reveals concentrations of synaptophysin in the perinuclear region. After neuronotypic differentiation, there is reduction in perinuclear labeling and concentration of label in swellings along PC12 cell processes. At the ultra-structural level, synaptophysin labeling is found on similar organelles in both undifferentiated and nerve growth factor-stimulated PC12 cells. Although the highest labeling densities were seen on small clear vesicles, specific labeling was also seen on dense core vesicles. The presence of synaptophysin on both small clear vesicles and dense core vesicles indicates potential functional similarities in these vesicle types. The changes in the levels and immunocytochemical distribution of synaptophysin after neuronotypic differentiation suggest possible functional heterogeneity among morphologically similar populations of small clear vesicles.