Immunoaffinity purification of the lipid transfer protein complex directly from human plasma

The human cholesteryl ester (CE) and triglyceride (TG) exchange protein (denoted LTC or lipid transfer complex) was isolated in a single step from plasma using immunoaffinity batch extraction. Antibodies were raised against two preparations of conventionally purified LTC. LTC-I and LTC-II (purified 20,000-fold and 3500-fold, respectively) were used as immunogens. The antiLTC antibodies were isolated by anion-exchange chromatography and coupled to Affi-Gel 10. Chromatography of plasma on antiLTC Affi-Gel removed all of the CE and TG transfer activity. Moreover, LTC prepared from both antiLTC-I and antiLTC-II-Affi-Gel matrices were identical when analyzed by sodium dodecyl sulfate-polyacrylamide gel LTC electrophoresis. LTC exhibited two protein bands of Mr (apparent) 67,000 and 58,000 and a broad, faintly staining region at greater than 150,000. Analysis of LTC by immunoblotting indicated that both antiLTC-I and antiLTC-II antibodies recognized the same LTC proteins. Isoelectric focussing of LTC gave two pI values, 5.2 and 8.7. These data suggest that LTC is a complex of specific proteins and perhaps lipid. Specific CE and TG exchange activities of immunoaffinity-purified LTC were comparable, although the activities were low with respect to that of the antigen used to generate antiLTC-I. This is not due to contamination of LTC by albumin, lecithin:cholesterol acyltransferase, or apolipoproteins AI, AII, B, CIII, D, or E.     

Lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholine of guinea pig very low density lipoproteins and discoidal complexes of phospholipid and apolipoprotein: effect of apolipoprotein C-II on the catalytic mechanism

To elucidate the mechanism by which apolipoprotein C-II (apoC-II) enhances the activity of lipoprotein lipase (LpL), discoidal phospholipid complexes were prepared with apoC-III and di[(14)C]palmitoyl phosphatidylcholine (DPPC) and containing various amounts of apoC-II. The rate of DPPC hydrolysis catalyzed by purified bovine milk LpL was determined on the isolated complexes. The rate of hydrolysis was optimal at pH 8.0. Analysis of enzyme kinetic data over a range of phospholipid concentrations revealed that the major effect of apoC-II was to increase the maximal velocity (V(max)) some 50-fold with a limited effect on the Michaelis constant (K(m)). V(max) of the apoC-III complex containing no apoC-II was 9.2 nmol/min per mg LpL vs. 482 nmol/min per mg LpL for the complex containing only apoC-II. The effect of apoC-II on enzyme kinetic parameters for LpL-catalyzed hydrolysis of DPPC complexes was compared to that on the parameters for hydrolysis of DPPC and trioleoylglycerol incorporated into guinea pig very low density lipoproteins (VLDL(p)) which lack the equivalent of human apoC-II. Tri[(3)H]oleoylglycerol-labeled VLDL(p) were obtained by perfusion of guinea pig liver with [(3)H]oleic acid. Di[(14)C]palmitoyl phosphatidylcholine was incorporated into the VLDL(p) by incubation of VLDL(p) with sonicated vesicles of di[(14)C]palmitoyl phosphatidylcholine and purified bovine liver phosphatidylcholine exchange protein. The rates of LpL-catalyzed hydrolysis of trioleoylglycerol and DPPC were determined at pH 7.4 and 8.5 in the presence and absence of apoC-II. In the presence of apoC-II, the V(max) for DPPC hydrolysis in guinea pig VLDL(p) increased at both pH 7.4 and pH 8.5 (2.4- and 3.2-fold, respectively); the value of K(m) did not change at either pH (0.23 mm). On the other hand, the kinetic value of K(m) for triacylglycerol hydrolysis in the presence of apoC-II decreased at both pH 7.4 (3.05 vs. 0.54 mm) and pH 8.5 (2.73 vs. 0.62 mm). These kinetic studies suggest that apoC-II enhances phospholipid hydrolysis by LpL in apoC-III-DPPC discoidal complexes and VLDL(p) mainly by increasing the V(max) of the enzyme for the substrates, whereas the activator protein primarily causes a decrease in the apparent K(m) for triacylglycerol hydrolysis.-Shirai, K., T. J. Fitzharris, M. Shinomiya, H. G. Muntz, J. A. K. Harmony, R. L. Jackson and D. M. Quinn. Lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholine of guinea pig very low density lipoproteins and discoidal complexes of phospholipid and apolipoprotein: effect of apolipoprotein C-II on the catalytic mechanism.     

Accessory cells induce a polyphosphatidylinositol response when cultured with mitogen-activated T lymphocytes

Monocytes (MO) influenced phosphoinositide metabolism when human T lymphocytes, isolated from peripheral blood, were activated by polyclonal mitogens. In the 3 hr immediately following mitogenic challenge, the synthesis of phosphatidylinositol (PI) was augmented and the synthesis of PI-4-phosphate (PIP) and PI-4,5-bisphosphate (PIP2) was induced in cultures of T lymphocytes and MO. In addition, MO induced a rapid and transient degradation of PIP and PIP2 in T cells prelabeled with [32P]PL and subsequently activated by mitogen. Induction of a PIP/PIP2 response correlated well with induction of DNA replication by MO when T cells were activated by phytohemagglutinin or by neuraminidase plus galactose oxidase. MO did not influence polyphosphoinositide metabolism when T cells were stimulated by the nonmitogenic lectin wheat germ agglutinin. Interleukin 1 could not substitute for monocytes in inducing a polyphosphoinositide response. By causing a rapid and transient release of the second messengers diacylglycerol and inositol phosphates and by subsequently increasing their cellular precursors, MO may induce the interleukin 2 responsive state in T lymphocytes.     

Interaction of human plasma low density lipoprotein with concanavalin A and with ricin.

Native human plasma low density lipoprotein (LDL) interacts with concanavalin A but not with ricin; apOLDL reacts with both lectins. Each reaction is inhibited by the appropriate lectin-specific carbohydrate. The "receptors" on LDL for these two lectins are not destroyed by digestion by proteolytic enzymes. Peptide hydrolysis does not influence the reactivity of LDL toward concanavalin A. It does, however, substantially enhance the ability of the lipoprotein to interact with ricin. The data strongly suggest that the carbohydrate protion of a glycoprotein component of LDL is bound at the saccharidespecific active site on the lectin.     

Visuospatial Working Memory in Toddlers with a History of Periventricular Leukomalacia: An EEG Narrow-Band Power Analysis

Background

Periventricular Leukomalacia (PVL) affects white matter, but grey matter injuries have also been reported, particularly in the dorsomedial nucleus and the cortex. Both structures have been related to working memory (WM) processes. The aim of this study was to compare behavioral performances and EEG power spectra during a visuospatial working memory task (VSWMT) of toddlers with a history of PVL and healthy toddlers.

Methodology/Principal Findings

A prospective, comparative study of WM was conducted in toddlers with a history of PVL and healthy toddlers. The task responses and the EEG narrow-band power spectra during a VSWMT were compared in both groups. The EEG absolute power was analyzed during the following three conditions: baseline, attention and WM retention. The number of correct responses was higher in the healthy group (20.5±5.0) compared to the PVL group (16.1±3.9) (p = 0.04). The healthy group had absolute power EEG increases (p≤0.05) during WM compared to the attention condition in the bilateral frontal and right temporal, parietal and occipital regions in frequencies ranging from 1.17 to 2.34 Hz and in the right temporal, parietal and occipital regions in frequencies ranging from 14.06 to 15.23 Hz. In contrast, the PVL group had absolute power increases (p≤0.05) in the bilateral fronto-parietal, left central and occipital regions in frequencies that ranged from 1.17 to 3.52 Hz and in the bilateral frontal and right temporal regions in frequencies ranging from 9.37 to 19.14 Hz.

Conclusions/Significance

This study provides evidence that PVL toddlers have visuospatial WM deficits and a very different pattern of absolute power increases compared to a healthy group of toddlers, with greater absolute power in the low frequency range and widespread neuronal networks in the WM retention phase.     

ApoE and clusterin cooperatively suppress Abeta levels and deposition: evidence that ApoE regulates extracellular Abeta metabolism in vivo

Apolipoprotein E (apoE) and clusterin can influence structure, toxicity, and accumulation of the amyloid-beta (Abeta) peptide in brain. Both molecules may also be involved in Abeta metabolism prior to its deposition. To assess this possibility, we compared PDAPP transgenic mice that develop age-dependent Abeta accumulation in the absence of apoE or clusterin as well as in the absence of both proteins. apoE(-/-) and clusterin(-/-) mice accumulated similar Abeta levels but much less fibrillar Abeta. In contrast, apoE(-/-)/clusterin(-/-) mice had both earlier onset and markedly increased Abeta and amyloid deposition. Both apoE(-/-) and apoE(-/-)/clusterin(-/-) mice had elevated CSF and brain interstitial fluid Abeta, as well as significant differences in the elimination half-life of interstitial fluid Abeta measured by in vivo microdialysis. These findings demonstrate additive effects of apoE and clusterin on influencing Abeta deposition and that apoE plays an important role in regulating extracellular CNS Abeta metabolism independent of Abeta synthesis.     

Clusterin contributes to caspase-3-independent brain injury following neonatal hypoxia-ischemia

Clusterin, also known as apolipoprotein J, is a ubiquitously expressed molecule thought to influence a variety of processes including cell death. In the brain, it accumulates in dying neurons following seizures and hypoxic-ischemic (H-I) injury. Despite this, in vivo evidence that clusterin directly influences cell death is lacking. Following neonatal H-I brain injury in mice (a model of cerebral palsy), there was evidence of apoptotic changes (neuronal caspase-3 activation), as well as accumulation of clusterin in dying neurons. Clusterin-deficient mice had 50% less brain injury following neonatal H-I. Surprisingly, the absence of clusterin had no effect on caspase-3 activation, and clusterin accumulation and caspase-3 activation did not colocalize to the same cells. Studies with cultured cortical neurons demonstrated that exogenous purified astrocyte-secreted clusterin exacerbated oxygen/glucose-deprivation-induced necrotic death. These results indicate that clusterin may be a new therapeutic target to modulate non-caspase-dependent neuronal death following acute brain injury.