Development of capture assays for different modifications of human low-density lipoprotein

Antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL), copper-oxidized LDL (oxLDL), Nepsilon(carboxymethyl) lysine (CML)-modified LDL, and advanced glycosylation end product (AGE)-modified LDL were obtained by immunization of rabbits with in vitro-modified human LDL preparations. After absorption of apolipoprotein B (ApoB) antibodies, we obtained antibodies specific for each modified lipoprotein with unique patterns of reactivity. MDA-LDL antibodies reacted strongly with MDA-LDL and also with oxLDL. CML-LDL antibodies reacted strongly with CML-LDL and also AGE-LDL. oxLDL antibodies reacted with oxLDL but not with MDA-LDL, and AGE-LDL antibodies reacted with AGE-LDL but not with CML-LDL. Capture assays were set with each antiserum, and we tested their ability to capture ApoB-containing lipoproteins isolated from precipitated immune complexes (IC) and from the supernatants remaining after IC precipitation (free lipoproteins). All antibodies captured lipoproteins contained in IC more effectively than free lipoproteins. Analysis of lipoproteins in IC by gas chromatography-mass spectrometry showed that they contained MDA-LDL and CML-LDL in significantly higher concentrations than free lipoproteins. A significant correlation (r=0.706, P<0.019) was obtained between the MDA concentrations determined by chemical analysis and by the capture assay of lipoproteins present in IC. In conclusion, we have developed capture assays for different LDL modifications in human ApoB/E lipoprotein-rich fractions isolated from precipitated IC. This approach obviates the interference of IC in previously reported modified LDL assays and allows determination of the degree of modification of LDL with greater accuracy.     

Isolation of a new clathrin heavy chain gene with muscle-specific expression from the region commonly deleted in velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are developmental disorders characterized by a spectrum of phenotypes including velopharyngeal insufficiency, conotruncal heart defects and facial dysmorphology among others. Eighty to eighty-five percent of VCFS/DGS patients are hemizygous for a portion of chromosome 22. It is likely that the genes encoded by this region play a role in the etiology of the phenotypes associated with the disorders. Using a cDNA selection protocol, we isolated a novel clathrin heavy chain cDNA (CLTD) from the VCFS/DGS minimally deleted interval. The cDNA encodes a protein of 1638 amino acids. CLTD shares significant homology, but is not identical to the ubiquitously expressed clathrin heavy chain gene. The CLTD gene also shows a unique pattern of expression, having its maximal level of expression in skeletal muscle. Velopharyngeal insufficiency and muscle weakness are common features of VCFS patients. Based on the location and expression pattern of CLTD, we suggest hemizygosity at this locus may play a role in the etiology of one of the VCFS-associated phenotypes.      

Argyrophilic cells in 202 human mucinous breast carcinomas. Relation to histopathologic and clinical factors

Two hundred two human, mucinous breast carcinomas were investigated for the presence of argyrophilic granules, and these granules were found in 25% of the cases. The granules were located in the cytoplasm and were heterogeneously distributed within the tumors. Tumors with granules were otherwise morphologically indistinguishable from those tumors without granules. The recurrence-free survival was independent of the presence of granules, and no relation was found to other clinical or histopathologic factors. Tumors with granules were found to be estrogen-receptor positive, and they appear to have a slightly less aggressive growth pattern than tumors without granules, but the difference is far from being statistically significant. It is concluded that there is no convincing evidence that this group of primary breast carcinomas with argyrophilia originates from APUD cells.     

Inhibition of host resistance by nutritional hypercholesteremia.

Previous experiments showed that nutritionally induced hypercholesteremia in mice caused an increase in susceptibility to coxsackievirus B, with a marked suppression of cellular infiltrates in infected tissues and an increased mortality. The present studies demonstrated that a hypercholesteremic diet was associated with an inhibition in host resistance as measured by susceptibility to Listeria monocytogenes infection and the growth of two transplanted syngeneic murine tumors. Moreover, the ability of Corynebacterium parvum to induce regression of a transplanted methylcholanthrene-induced fibrosarcoma was inhibited in hypercholesteremic hosts, as was the histiocytic infiltration normally accompanying C. parvum inoculation. In contrast, the peritoneal macrophages from C. parvum-treated hypercholesteremic mice were indistinguishable from similarly treated macrophages from normal mice with respect to their in vitro tumoricidal activity and the presence of a cell surface antigen associated with activated macrophages. Hypercholesteremia was also associated with a decreased antibody response to sheep erythrocytes in vivo, but dit not appear to exert a detrimental effect on B- or T-cell blastogenesis when tested in vitro. The findings that the hypercholesteremic diet was associated with an impairment in the host immune response and increased susceptibility to viral, bacterial, and tumor cell challenge are discussed with respect to virus-lipid interactions in the pathogenesis of atherogenesis and diabetes mellitus.     

An enhanced chemiluminescent enzyme immunoassay for follicle stimulating hormone

An enhanced chemiluminescent enzyme immunoassay for serum follicle stimulating hormone is described which involves sequential reaction of anti-follicle stimulating hormone antibody immobilised to the inside surface of an opaque microtitre plate with sample, monoclonal anti-alpha thyroid stimulating hormone antibody, and an anti-mouse IgG - horseradish peroxidase conjungate. Bound peroxidase activity was measured using a p-hydroxycinnamic acid enhanced chemiluminescent luminol-hydrogen peroxide reaction. The assay was sensitive (detection limit 0.01 mU/well) precise (intra-assay precision 2.5-8.1%, inter-assay precision 6.7-11.9%) and results obtained with this assay and a competitive radioimmunoassay were in good agreement (correlation coefficient 0.98).     

Orexin A mediation of time spent moving in rats: Neural mechanisms

The brain regulates energy balance and spontaneous physical activity, including both small- and large-motor activities. Neural mediators of spontaneous physical activity are currently undefined, although the amount of time spent in sedentary positions versus standing and ambulating may be important in the energetics of human obesity. Orexin A, a neuropeptide produced in caudal hypothalamic areas and projecting throughout the neuraxis, enhances arousal and spontaneous physical activity. To test the hypothesis that orexin A affects the amount of time spent moving, we injected orexin A (0–1000 pmol) into three orexin projection sites in male Sprague–Dawley rats: hypothalamic paraventricular nucleus, rostral lateral hypothalamic area and substantia nigra pars compacta, and measured spontaneous physical activity. Orexin A affects local GABA release and we co-injected orexin A with a GABA agonist, muscimol, in each brain site. Dopamine signaling is important to substantia nigra function and so we also co-injected a dopamine 1 receptor antagonist (SCH 23390) in the substantia nigra pars compacta. In all brain sites orexin A significantly increased time spent vertical and ambulating. Muscimol significantly and dose-dependently inhibited orexin A effects on time spent moving only when administered to the rostral lateral hypothalamic area. In the substantia nigra pars compacta, SCH 23390 completely blocked orexin A–induced ambulation. These data indicate that orexin A influences time spent moving, in three brain sites utilizing separate signaling mechanisms. That orexin A modulation of spontaneous physical activity occurs in brain areas with multiple roles indicates generalization across brain site, and may reflect a fundamental mechanism for enhancing activity levels. This potential for conferring physical activity stimulation may be useful for inducing shifts in time spent moving, which has important implications for obesity.     

An improved immunoblotting procedure for the detection of antibodies against HIV

Immunoblotting ('Western blotting') is routinely used for detection of antibodies against HIV in the diagnosis of HIV infection. We describe an improved procedure, which does not require virus purification and is easy to control for 'false-positive' results. The technique also does not produce erroneous results due to reactivity of the developing system with residual cellular proteins or viral antigens and does not give high nonspecific background staining. The technique can be applied to the detection of antibodies to HIV in serum, plasma, and blood products.