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Antimetabolites have proven successful as therapeutics for advanced-stage breast cancers, but are often accompanied by severe side effects that can limit treatment regimens. 5-Fluorouracil (5-FU), an antimetabolite that inhibits cell proliferation, has served an important role in standard chemotherapy protocols for a variety of solid tumors. Although reasonable response rates have been reported for 5-FU, continued exploration is necessary to improve clinical outcomes and reduce cytotoxic side effects that are an inherent problem for chemotherapeutic interventions. Because of its diverse anticancer properties, we explored whether by combining the natural product curcumin with 5-FU, synergistic improvements in preventing breast cancer cell proliferation and/or provide protection against 5-FU-induced cytotoxicity could be achieved. Indeed both curcumin and 5-FU inhibit DNA synthesis in MDA-MB-231 cells using BrdU incorporation assays; however, combined treatment showed no synergistic improvement. We next established the cytotoxicity profile for 5-FU in MDA-MB-231 cells using a tetrazolium-based cell viability assay and obtained an LD50 value of 28 μM. When 5-FU incubations were repeated with the addition of curcumin, the LD50 value increased to 200-300 μM, representing a 7-10-fold protection by curcumin against 5-FU cytotoxicity. These findings suggest that the addition of curcumin as an adjuvant therapy during 5-FU treatment might enhance the chemotherapeutic effectiveness of 5-FU by protecting normal cells from reduced viability and thus permitting higher dosing or longer treatment times. This would be especially important to those individuals who are plagued with severe cytotoxicities and require frequent interruptions, or even early termination of their treatment regimens.

It is generally accepted that the selection of gene variants during human evolution optimized energy metabolism that now interacts with our obesogenic environment to increase the prevalence of obesity. The purpose of this study was to perform a global evolutionary and metabolic analysis of human obesity gene risk variants (110 human obesity genes with 127 nearest gene risk variants) identified using genome-wide association studies (GWAS) to enhance our knowledge of early and late genotypes. As a result of determining the mean frequency of these obesity gene risk variants in 13 available populations from around the world our results provide evidence for the early selection of ancestral risk variants (defined as selection before migration from Africa) and late selection of derived risk variants (defined as selection after migration from Africa). Our results also provide novel information for association of these obesity genes or encoded proteins with diverse metabolic pathways and other human diseases. The overall results indicate a significant differential evolutionary pattern for the selection of obesity gene ancestral and derived risk variants proposed to optimize energy metabolism in varying global environments and complex association with metabolic pathways and other human diseases. These results are consistent with obesity genes that encode proteins possessing a fundamental role in maintaining energy metabolism and survival during the course of human evolution.

The transcription factor, Nuclear Factor-kappaB (NF-kappaB), regulates many genes involved in host immunity and cell survival. Unregulated NF-kappaB activity has been linked to many chronic inflammatory diseases and is an important target for the identification of inhibitors to better manage these disorders. We present a novel screening system to identify NF-kappaB inhibitors that combines sensitive fluorescence detection with medium- to high-throughput flow cytometry (HyperCyt). To validate this approach, we quantified the activation of NF-kappaB by standard flow cytometry and the HyperCyt platform. Results were comparable with regard to EC(50) values for TNFalpha-mediated activation; however, the HyperCyt platform provided more sensitive signal detection and a greater linear range for detection. To demonstrate the usefulness of this screening tool, we identified a novel inhibitor of NF-kappaB activation from a resveratrol-based chemical library. The inhibition of NF-kappaB activation by analog 6q (IC(50) = 19 microm) showed a 3.7-fold improvement over that of resveratrol (IC(50) approximately 70 microm).

Background We evaluated the relationship between an early inflammatory biomarker, chemokine (C-C motif) ligand 2 (CCL2), and other clinical biomarkers and lifestyle behaviors, in overweight/obese adolescents at high risk of developing cardiometabolic derangements. Methods We collected anthropometric measurements, clinical biomarkers, and three 24-h dietary recalls from 21 vocational high school students (91% male), 14-19 years, with body mass index (BMI) ≥25 kg/m2. Pearson's or Spearman's correlation coefficients were used to examine relationships. Results Mean BMI was 33.2 kg/m2 (range 25.7-45.6) and 38% were prediabetic by fasting glucose. Mean CCL2 was 512.9 pg/mL (range 220-917) and positively correlated with triglycerides (r=0.45; p=0.04) and TNF-α (r=0.57; p=0.007) and marginally negatively correlated with fruit/vegetable intake (r=-0.42, p=0.06) and omega-3 fatty acids (r=-0.41, p=0.07). Conclusions CCL2 was positively associated with pro-inflammatory biomarkers and negatively associated with some anti-inflammatory dietary factors.

Low-density lipoprotein receptor-related protein (LRP) mediates internalization of urokinase:plasminogen activator inhibitor complexes (uPA:PAI-1) and the urokinase receptor (uPAR). Here we investigated whether direct interaction between uPAR, a glycosyl-phosphatidylinositol–anchored protein, and LRP, a transmembrane receptor, is required for clearance of uPA:PAI-1, regeneration of unoccupied uPAR, activation of plasminogen, and the ability of HT1080 cells to invade extracellular matrix. We found that in the absence of uPA:PAI-1, uPAR is randomly distributed along the plasma membrane, whereas uPA:PAI-1 promotes formation of uPAR-LRP complexes and initiates redistribution of occupied uPAR to clathrin-coated pits. uPAR-LRP complexes are endocytosed via clathrin-coated vesicles and traffic together to early endosomes (EE) because they can be coimmunoprecipitated from immunoisolated EE, and internalization is blocked by depletion of intracellular K+. Direct binding of domain 3 (D3) of uPAR to LRP is required for clearance of uPA-PAI-1–occupied uPAR because internalization is blocked by incubation with recombinant D3. Moreover, uPA-dependent plasmin generation and the ability of HT1080 cells to migrate through Matrigel-coated invasion chambers are also inhibited in the presence of D3. These results demonstrate that GPI-anchored uPAR is endocytosed by piggybacking on LRP and that direct binding of occupied uPAR to LRP is essential for internalization of occupied uPAR, regeneration of unoccupied uPAR, plasmin generation, and invasion and migration through extracellular matrix.

OBJECTIVE To explore the hypothesis that CD36, a scavenger receptor and fatty acid translocase, is upregulated in peripheral blood mononuclear cells (PBMCs) among patients with type 2 diabetes and is a biomarker of PBMC activation and inflammation. RESEARCH DESIGN AND METHODS We used a cross-sectional observational design to study a multi-racial/ethnic population sample consisting of Caucasians, Hispanics, and Native Americans with type 2 diabetes (n = 33) and nondiabetic control subjects (n = 27). PBMC CD36 mRNA/protein and plasma high sensitivity (hs) C-reactive protein (hsCRP), hs–interleukin-6 (hsIL-6), and adiponectin were measured. RESULTS Unadjusted PBMC CD36 mRNA and protein were 1.56- and 1.63-fold higher, respectively, among type 2 diabetic subjects versus control subjects. PBMC CD36 protein was directly associated with CD36 mRNA, plasma hsCRP, and hsIL-6 and inversely associated with plasma adiponectin in both groups. CONCLUSIONS Increased CD36 expression is a biomarker of PBMC activation and inflammation and may become a useful tool in cardiovascular disease risk stratification.

Ankyrin-repeat family A protein (ANKRA) is a novel protein that interacts directly and specifically with the cytoplasmic tail of megalin in the yeast two-hybrid system and glutathione-S-transferase pull-down assays. ANKRA has three ankyrin repeats and shows 61% overall homology to regulatory factor X, ankyrin repeat-containing protein. Mapping studies show that the three ankyrin repeats and C-terminus of ANKRA are required for binding to a unique juxtamembrane, 19-amino acid sequence on the megalin tail. Point mutational analysis reveals that a proline-rich motif (PXXPXXP) within this region is the site of ANKRA binding. ANKRA interacts with megalin but not with low-density lipoprotein receptor related protein, in keeping with the fact that the sequence of the megalin tail is unique. By cell fractionation, ANKRA is found both in the cytosol and associated with membranes enriched in megalin in L2 cells and proximal tubule cells. By immunofluorescence, ANKRA is concentrated near megalin along the plasma membrane of L2 cells and in the kidney cortex is expressed in glomerular and proximal tubule epithelia which also express megalin. These observations suggest that ANKRA may play a unique role in megalin's function as a clearance receptor in the kidney and L2 cells. In addition, ANKRA may have other partners because northern blot analysis reveals that ANKRA is more broadly expressed than megalin, and by immunofluorescence ANKRA is also expressed in connecting tubule cells and principal cells of collecting ducts.

The receptor-associated protein (RAP) is a chaperone found primarily in the endoplasmic reticulum (ER) that plays a necessary role in the folding and exocytic trafficking of members of the LDL receptor gene family including megalin and the LDL receptor-related protein (LRP). Recently, RAP has been shown to interact with a growing number of proteins including several that are unrelated to the LDL receptor family as well as new members of this rapidly expanding family. Based on these observations, we have applied chemical crosslinking procedures to identify additional novel RAP-interacting proteins, and thereby better characterize the scope of RAP's ER-related function. In this study, we have identified eight proteins with molecular weights of 32, 35, 46, 55, 70, 95, 170, and 200 kDa that interact with endogenous RAP. These proteins were found to associate with RAP in multiple cell types from different species, suggesting that their expression and interactions with RAP are ubiquitous. Results of pulse-chase experiments show that most of the proteins remain sensitive to endoglycosidase-H digestion, and also remain stably associated with RAP over an extended period, suggesting that they are ER resident proteins. All of the RAP-associated proteins appear to be largely soluble as they partition into the aqueous phase following TX-114 detergent extraction. Sequence analysis and immunoblotting of the 46-kDa RAP-associated glycoprotein (gp46) shows that it is structurally and immunologically related to actin. If gp46 is also functionally related to actin as an intracellular structural protein, it may represent a novel component of the putative ER matrix.

Podocalyxin is a major membrane protein of the glomerular epithelium and is thought to be involved in maintenance of the architecture of the foot processes and filtration slits characteristic of this unique epithelium by virtue of its high negative charge. However, until now there has been no direct evidence for podocalyxin's function. Podocalyxin is a type 1 transmembrane sialoprotein with an N-terminal mucin-like domain. To assess its function, we cloned rat podocalyxin and examined the effects of its expression on the cell adhesion properties of stably transfected Chinese hamster ovary (CHO)-K1 and Madin-Darby canine kidney (MDCK) cells and inducible ecdysone receptor–expressing (EcR)-CHO cells. In a cell aggregation assay, CHO-K1 cells expressing high levels of podocalyxin showed complete inhibition of cell aggregation, and MDCK transfectants showed greatly reduced aggregation (∼60–80%) compared with parental cells. In EcR-CHO cells, the expression level of podocalyxin induced by increasing levels of ecdysone analogue correlated closely with the antiadhesion effect. The inhibitory effect of podocalyxin was reversed by treatment of the cells with Arthrobacter ureafaciens sialidase, indicating that sialic acid is required for inhibition of cell adhesion. Overexpression of podocalyxin also affected transepithelial resistance and the distribution of junctional proteins in MDCK cells by an unknown mechanism that may involve interaction with the actin cytoskeleton. These results provide direct evidence that podocalyxin functions as an antiadhesin that maintains an open filtration pathway between neighboring foot processes in the glomerular epithelium by charge repulsion.

Megalin (gp330) is the main target antigen involved in the induction of Heymann nephritis (HN), a rat model of human membranous nephropathy. Its large extracellular region contains four putative ligand-binding domains separated by spacer regions. Previously, it was reported that the second ligand-binding domain (LBD II) of megalin is involved in the pathogenesis of passive HN because it is capable of binding antibodies in vivo and initiating formation of immune deposits (ID). This study explores the possibility that pathogenic epitopes might also be present in the other putative ligand-binding domains. Recombinant fragments of ligand-binding domains (LBD) I through IV expressed in a baculovirus system were used to generate polyclonal domain-specific antibodies. Antibodies raised against each of the recombinant megalin fragments reacted preferentially with its respective antigen and with whole megalin by immunoblotting. Each of the antibodies also gave a characteristic brush-border staining for megalin by indirect immunofluorescence on rat kidney. When rats were injected with the domain-specific antibodies to test their ability to produce passive HN, glomerular ID were present in kidneys of all injected animals. The staining pattern in glomeruli of rats injected with LBD I, III, or IV was similar to that obtained with antibodies to LBD II. It is concluded that passive HN can be induced with antibodies against LBD I, III, and IV, as well as LBD II, and that each of the ligand-binding domains contains a pathogenic epitope. These findings provide further evidence for the multiple epitope model of HN.

In our program to develop non-invasive magnetic resonance imaging (MRI) methods for the diagnosis of Alzheimer's disease (AD), we have synthesized antibody-conjugated, superparamagnetic iron oxide nanoparticles (SPIONs) for use as an in vivo agent for MRI detection of amyloid-β plaques in AD. Here we report studies in AβPP/PS1 transgenic mice, which demonstrate the ability of novel anti-AβPP conjugated SPIONs to penetrate the blood-brain barrier to act as a contrast agent for MR imaging of plaques. The conspicuity of the plaques increased from an average Z-score of 5.1 ± 0.5 to 8.3 ± 0.2 when the plaque contrast to noise ratio was compared in control AD mice with AD mice treated with SPIONs. The number of MRI-visible plaques per brain increased from 347 ± 45 in the control AD mice, to 668 ± 86 in the SPION treated mice. These results indicated that our SPION enhanced amyloid-β detection method delivers an efficacious, non-invasive MRI detection method in transgenic mice.

Amyloid-like plaques are characteristic lesions defining the neuropathology of Alzheimer's disease (AD). The size and density of these plaques are closely associated with cognitive decline. To combat this disease, the few therapies that are available rely on drugs that increase neurotransmission; however, this approach has had limited success as it has simply slowed an imminent decline and failed to target the root cause of AD. Amyloid-like deposits result from aggregation of the Aβ peptide, and thus, reducing amyloid burden by preventing Aβ aggregation represents an attractive approach to improve the therapeutic arsenal for AD. Recent studies have shown that the natural product curcumin is capable of crossing the blood-brain barrier in the CNS in sufficient quantities so as to reduce amyloid plaque burden. Based upon this bioactivity, we hypothesized that curcumin presents molecular features that make it an excellent lead compound for the development of more effective inhibitors of Aβ aggregation. To explore this hypothesis, we screened a library of curcumin analogs and identified structural features that contribute to the anti-oligomerization activity of curcumin and its analogs. First, at least one enone group in the spacer between aryl rings is necessary for measureable anti-Aβ aggregation activity. Second, an unsaturated carbon spacer between aryl rings is essential for inhibitory activity, as none of the saturated carbon spacers showed any margin of improvement over that of native curcumin. Third, methoxyl and hydroxyl substitutions in the meta- and para-positions on the aryl rings appear necessary for some measure of improved inhibitory activity. The best lead inhibitors have either their meta- and para-substituted methoxyl and hydroxyl groups reversed from that of curcumin or methoxyl or hydroxyl groups placed in both positions. The simple substitution of the para-hydroxy group on curcumin with a methoxy substitution improved inhibitor function by 6-7-fold over that measured for curcumin.

It is apparent that significant progress has been made in the characterization of gp330 in the years that have elapsed since its initial identification as the nephritogenic antigen of Heymann nephritis. However, there are still many gaps in our knowledge and we do not yet have a full picture of the molecular events leading to the formation of immune deposits in glomerular capillaries. Moreover, we still do not have direct information on the normal function(s) of gp330 and RAP and their trafficking in renal and other epithelia. The availability of the yolk sac and other cell lines that express gp330 and RAP together with the identification of the functional domains of RAP should greatly facilitate experimental studies designed to elucidate these problems. Progress will also be greatly facilitated in the future when the complete amino-acid sequence of gp330 becomes available, making possible further structural studies. It is our hope that new knowledge obtained on the molecular mechanisms of HN will provide insights into the molecular pathogenesis of human membranous nephropathy and will provide a strategy for the design of appropriate treatments to interrupt the process.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a primary choice of therapy for diseases with a chronic inflammatory component. Unfortunately, long-term NSAID therapy is often accompanied by severe side effects, including cardiovascular and gastrointestinal complications. Because of this, there is critical need for identification of new and safer treatments for chronic inflammation to circumvent these side effects. Inflammatory diseases have been successfully remedied with natural herbs by many cultures. To better understand the potential of natural herbs in treating chronic inflammation and to identify their mechanism of action, we have evaluated the anti-inflammatory activities of 20 medicinal herbs commonly used in the Hispanic culture. We have established a standardized method for preparing aqueous extracts (teas) from the selected medicinal herbs and screened for inhibition of tumor necrosis factor-alpha-induced activation of nuclear factor kappaB (NF-kappaB), which is the central signaling pathway of the inflammatory response. A number of herbal teas were identified that exhibited significant anti-inflammatory activity. In particular, tea from the herb commonly called laurel was found to be an especially potent inhibitor of NF-kappaB-dependent cyclooxygenase-2 gene expression and prostaglandin E(2) production in cultured murine macrophages. These findings indicate that laurel tea extract contains potent anti-inflammatory compounds that function by inhibiting the major signal transduction pathway responsible for inducing an inflammatory event. Based on these results, laurel may represent a new, safe therapeutic agent for managing chronic inflammation.

Background The activator protein-1 (AP-1) family of transcription factors contributes to regulation of numerous genes involved in proliferation, apoptosis, and tumorigenesis. A wide array of stimuli can activate AP-1, including pro-inflammatory cytokines, growth factors, tumor promoters and stress. Numerous plant polyphenols have been shown to inhibit the activation of AP-1, which often is ascribed to the anti-oxidant properties of these natural products. Methods In the present study, a library of substituted trans-stilbenes, including polyphenols, was screened for activity against the TPA-induced activation of AP-1 using the Panomics AP-1 Reporter 293 Stable Cell Line, which is designed for screening potential inhibitors or activators. Results Several trans-stilbenes were identified that inhibit TPA-induced activation of AP-1, with IC50 values as low as 0.5 μM. Moreover, some other trans-stilbenes were able to enhance the effects of TPA 2 to 3-fold. Many of the trans-stilbenes identified as inhibitors or enhancers are devoid of anti-oxidant properties. Conclusion The ability of trans-stilbenes to inhibit or enhance the effects of TPA does not depend upon their anti-oxidant properties.

The transcription factor nuclear factor kappaB (NF-kappaB), which regulates expression of numerous antiinflammatory genes as well as genes that promote development of the prosurvival, antiapoptotic state is up-regulated in many cancer cells. The natural product resveratrol, a polyphenolic trans-stilbene, has numerous biological activities and is a known inhibitor of activation of NF-kappaB, which may account for some of its biological activities. Resveratrol exhibits activity against a wide variety of cancer cells and has demonstrated activity as a cancer chemopreventive against all stages, i.e., initiation, promotion, and progression. The biological activities of resveratrol are often ascribed to its antioxidant activity. Both antioxidant activity and biological activities of analogues of resveratrol depend upon the number and location of the hydroxy groups. In the present study, phenolic analogues of resveratrol and a series of substituted trans-stilbenes without hydroxy groups were compared with resveratrol for their abilities to inhibit the human tumor necrosis factor alpha-induced (TNF-alpha) activation of NF-kappaB, using the Panomics NF-kappaB stable reporter cell line 293/NF-kappaB-luc. A series of 75 compounds was screened to identify substituted trans-stilbenes that were more active than resveratrol. Dose-response studies of the most active compounds were carried out to obtain IC50 values. Numerous compounds were identified that were more active than resveratrol, including compounds that were devoid of hydroxy groups and were 100-fold more potent than resveratrol. The substituted trans-stilbenes that were potent inhibitors of the activation of NFkappaB generally did not exhibit antioxidant activity. The results from screening were confirmed using BV-2 microglial cells where resveratrol and analogues were shown to inhibit LPS-induced COX-2 expression.

Background Triacylglyerol-rich very low density lipoprotein (VLDL) particles are the primary carriers of fatty acids in the circulation and as such serve as a rich energy source for peripheral tissues. Receptor-mediated uptake of these particles is dependent upon prior association with apolipoprotein E (apoE-VLDL) and is brought about by cell surface heparan sulfate proteoglycans (HSPG) in some cell types and by the low density lipoprotein receptor-related protein (LRP) in others. Although LRP's role in apoE-VLDL uptake has been well studied, the identity of the HSPG family member that mediates apoE-VLDL uptake has not been established. We investigated if syndecan-1 (Syn-1), a transmembrane cell surface HSPG, is able to mediate the internalization of apoE-VLDL and examined the relationship between Syn-1 and LRP toward apoE-VLDL uptake. For this study, we used a human fibroblast cell line (GM00701) that expresses large amounts of LRP, but possesses no LDL receptor activity to eliminate its contributions toward apoE-VLDL uptake. Results Although LRP in these cells is fully active as established by substantial α2macroglobulin binding and internalization, uptake of apoE-VLDL is absent. Expression of human Syn-1 cDNA restored apoE-VLDL binding and uptake by these cells. Competition for this uptake with an LRP ligand-binding antagonist had little or no effect, whereas co-incubation with heparin abolished apoE-VLDL internalization. Depleting Syn-1 expressing cells of K+, to block clathrin-mediated endocytosis, showed no inhibition of Syn-1 internalization of apoE-VLDL. By contrast, treatment of cells with nystatin to inhibit lipid raft function, prevented the uptake of apoE-VLDL by Syn-1. Conclusion These data demonstrate that Syn-1 is able to mediate apoE-VLDL uptake in human fibroblasts with little or no contribution from LRP and that the endocytic path taken by Syn-1 is clathrin-independent and relies upon lipid raft function. These data are consistent with previous studies demonstrating Syn-1 association with lipid raft domains.

The activator protein-1 (AP-1) family of transcription factors, including the most common member c-Jun-c-Fos, participates in regulation of expression of numerous genes involved in proliferation, apoptosis, and tumorigenesis in response to a wide array of stimuli including pro-inflammatory cytokines, growth factors, stress, and tumor promoters. A number of plant polyphenols including curcumin, a yellow compound in the spice turmeric, have been shown to inhibit the activation of AP-1. Curcumin is a polyphenolic dienone that is potentially reactive as a Michael acceptor and also is a strong anti-oxidant. Multiple activities reported for curcumin, including inhibition of the stress-induced activation of AP-1, have been suggested to involve the anti-oxidant properties of curcumin. In the present study, a library of analogs of curcumin was screened for activity against the TPA-induced activation of AP-1 using the Panomics AP-1 Reporter 293 stable cell line which is designed for screening potential inhibitors. Numerous analogs were identified that were more active than curcumin, including analogs that were not anti-oxidants and analogs that were not Michael acceptors. Clearly, anti-oxidant activity or reactivity as a Michael acceptor is not an essential feature of active compounds. In addition, a number of analogs were identified that enhanced the TPA-induced activation of AP-1. The results from screening were confirmed using BV-2 microglial cells where curcumin and analogs were shown to inhibit LPS-induced COX-2 expression; analogs identified as more potent than curcumin in the screening assay were also more potent than curcumin in preventing COX-2 expression.

In order to delineate the neuroprotective role of the low density lipoprotein receptor-related protein (LRP) against amyloid beta-protein toxicity, studies were performed in C6 cells challenged with amyloid beta-protein in the presence or absence of activated alpha(2)-macroglobulin. Toxicity was assessed via two cell viability assays. We found that this endocytic receptor conferred protection against amyloid beta-protein toxicity in the presence of activated alpha(2)-macroglobulin and its down-regulation via inhibition by receptor-associated protein or transfection of cells with presenilin 1, increased susceptibility to amyloid beta-protein toxicity. Increased surface LRP immunoreactivity in response to amyloid beta-protein challenge was associated with increased translocation of LRP from the endoplasmic reticulum to the surface, rather than from increased mRNA or protein expression. Furthermore, this translocation of LRP to the surface was mediated by a calcium/calmodulin protein kinase II-dependent signaling pathway. These studies provide evidence for a protective role of LRP against amyloid beta-protein toxicity and may explain the aggressive nature of presenilin-1 mutation in familial Alzheimer's disease.

Low density lipoprotein receptor-related protein (LRP) polymorphisms have recently been associated with an increased susceptibility of Alzheimer's disease (AD). Furthermore, LRP has been linked to molecules that confer susceptibility to AD (apolipoprotein E, alpha-2-macroglobulin, amyloid precursor protein), previously with the exception of the presenilins. Here we report that aberrant presenilin-1 expression in vivo and in vitro downregulates LRP. Specifically, transgenic mice overexpressing the M146L or L286V presenilin-1 mutation show decreased levels of LRP expression in neuronal populations where presenilin-1 and LRP are closely colocalized or coexpressed. Moreover, cell lines transfected with presenilin-1 also expressed decreased levels of LRP. These findings suggest that LRP may be central to AD pathogenesis since all proteins genetically associated with AD can now be linked via a single pathway to LRP.

Receptor-associated protein appears to play an important role in low-density lipoprotein receptor-related protein trafficking. Since ligands for the low-density lipoprotein receptor-related protein have been implicated in Alzheimer's disease and normal functioning of this protein is indispensable for central nervous system development, deficient receptor-associated protein expression may result in central nervous system alterations. In this study, receptor-associated protein knockout mice were behaviorally tested and nervous system integrity was assessed via in situ hybridization and immunocytochemical/laser confocal microscopy methods. Receptor-associated protein knockout mice were found to be cognitively impaired in the Morris water maze compared to controls. In wild-type mice, the receptor-associated protein was found to be highly co-expressed with somatostatin in hippocampal and neocortical inhibitory neurons. Receptor-associated protein knockout mice, however, showed a significant decrease in number of somatostatin-expressing neurons of the CA1 region and somatostatin expression within these neurons. The decreased number of somatostatin neurons significantly correlated with cognitive impairment observed in the receptor-associated protein knockout mice. These results suggest a novel role for receptor-associated protein in modulating the functioning of somatostatin-producing neurons. Furthermore, this has implications for Alzheimer's disease pathogenesis, in which altered regulation of both somatostatin and the known low-density lipoprotein receptor-related protein ligands are a consistent finding.

Heymann nephritis (HN) has been extensively studied as a model of human membranous nephropathy since it was first described by Heymann in 1959. HN was induced in active form by the immunization of rats with antigens derived from the proximal tubule brush border, resulting in subepithelial glomerular immune deposits. HN was also induced passively by the injection of antibrush border antibodies into normal rats. A breakthrough in the understanding of the pathogenesis of HN was made in the 1970s, when it was established that the disease was due to the binding of circulating antibodies to glomerular components. This in turn led to a search to identify the endogenous antigen(s). In 1982, gp330 (now called megalin), a glycoprotein located in clathrin-coated pits of glomerular and proximal tubular epithelia, was identified as a target antigen. In 1990, a second protein (44 kd), now known as RAP (for receptor associated protein), that binds to megalin was also shown to be a target antigen. Both molecules have been cloned and sequenced, and their role in normal epithelial cells has been explored. It has come to light that megalin (gp330) is a member of the low-density lipoprotein receptor gene family and functions as a multiligand receptor for the uptake of a variety of macromolecules (plasminogen, protease: protease inhibitor complexes, apolipoprotein E-enriched very low-density lipoproteins, lactoferrin, among others). RAP associates with megalin and appears to function as a chaperone assisting in the folding of megalin in the endoplasmic reticulum and its transport to the cell surface. This review considers what is now known about the structure, function, and trafficking of megalin and RAP and the role of these two molecules in the pathogenesis of HN.

The murmur produced by acute graded constriction of the base of the pulmonary artery was studied in the anesthetized dog by means of intraluminal phonocardiograms recorded from the pulmonary artery. In addition, right ventricular and distal pulmonary artery pressures were recorded, and the cardiac output was determined for each degree of pulmonary artery stenosis utilized in the study. A small diamond-shaped midsystolic murmur is normally recorded from inside the pulmonary artery. This murmur increases in intensity and duration with constriction of the base of the artery. A constant relationship was not found between the degree of pulmonary stenosis or pulmonary flow and the intensity of the murmur. The intensity did correlate, in an essentially linear fashion, with the average velocity of flow through the constricted area. The timing of the peak intensity of the murmur did not change in any consistent fashion with increasing degrees of pulmonary artery stenosis. Cardiac output and the velocity of flow through the constricted area of the pulmonary artery tended to increase with moderate degrees of constriction. When the lumen of the pulmonary artery was reduced 40 to 45 per cent, cardiac output and pulmonary artery pressure fell below control levels. With further constriction, cardiac output and pulmonary flow decreased; however, the velocity of flow through the stenotic area remained relatively rapid.

CLINICAL evidence indicates that adverse reactions to halothane are probably due to individual idiosyncrasies or hypersensitivities.1 2 3 4 5 Despite widespread use of the anesthetic, a recent nati...