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Megalin is the most abundant endocytic receptor in the proximal tubule epithelium (PTE), where it is concentrated in clathrin-coated pits (CCPs) and vesicles in the brush border region. The heterotrimeric G protein alpha subunit, Gαi3, has also been localized to the brush border region of PTE. By immunofluorescence GIPC and GAIP, components of G protein-mediated signaling pathways, are also concentrated in the brush border region of PTE and are present in megalin-expressing cell lines. By cell fractionation, these signaling molecules cosediment with megalin in brush border and microvillar fractions. GAIP is found by immunoelectron microscopy in CCPs, and GIPC is found in CCPs and apical tubules of endocytic compartments in the renal brush border. In precipitation assays, GST-GIPC specifically binds megalin. The concentration of Gαi3, GIPC, and GAIP with megalin in endocytic compartments of the proximal tubule, where extensive endocytosis occurs, and the interaction between GIPC and the cytoplasmic tail of megalin suggest a model whereby G protein-mediated signaling may regulate megalin’s endocytic function and/or trafficking.

Obesity often leads to increased systemic inflammation which is now thought to play a causative role in the development of atherosclerotic disease and insulin resistance. This inflammatory response originates within large adipose tissue depots and is initiated by classically activated macrophages that infiltrate the tissue from the circulation. The large number of macrophages residing in obese adipose tissue leads to significant increases in interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) secretion; achieving levels sufficient to elevate circulating plasma concentrations. These cytokines activate potent signals to initiate lipolysis, to release free fatty acids from triacylglycerol stores and contribute to hyperlipidemia in obese individuals. Obese adipose tissue responds to normal β-adrenergic and glucagon stimuli to recover from negative energy balance by inducing lipolysis. However, it is not clear what quantitative influence additional lipolytic stimulation by IL-6 and TNFα has on normal β-adrenergic activity. Although, β-adrenergic and cytokine signaling activate separate pathways for lipolytic activation, it is undefined whether the effects of multiple signaling events on lipolysis are additive or coincident. To clarify this issue, we measured lipolytic activity in 3T3-L1-derived adipocytes stimulated by a β-adrenergic agonist (isoproterenol), IL-6 or TNFα individually and in combinations as co- and tri-stimulation. Treatment of adipocytes with isoproterenol and either IL-6 or TNFα as co-stimulants increased lipolytic activation by approximately the sum of the individual ligands, suggesting contributions from two independent pathways. Co-stimulation with IL-6 and TNFα provided slightly more than an additive response indicating signaling contributions from independent and common pathways. Tri-stimulation resulted in the largest level of lipolytic activation with a value approximate to adding isoproterenol stimulation to a combined treatment of IL-6 and TNFα. The additive nature of cytokine signaling to β-adrenergic activity suggests its therapeutic inhibition will prevent excessive lipolysis, yet minimally interfere with maintaining normal responses to varying energy demands.

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.

Renal clearance is a major pathway for regulating the levels of insulin and other low molecular weight polypeptide hormones in the systemic circulation. Previous studies have shown that the reabsorption of insulin from the glomerular filtrate occurs by binding to as yet unidentified sites on the luminal surface of proximal tubule cells followed by endocytosis and degradation in lysosomes. In this study, an insulin binding site was identified in renal microvillar membranes by chemical cross-linking procedures. By immunoprecipitation it was demonstrated that this binding site is megalin, the large multiligand binding endocytic receptor that is abundantly expressed in clathrin-coated pits on the apical surface of proximal tubule cells. Moreover, using cytochemical procedures, it was also shown that megalin is able to internalize insulin into endocytic vesicles. In ligand blotting assays, megalin also bound several other low molecular weight polypeptides, including beta2-microglobulin, epidermal growth factor, prolactin, lysozyme, and cytochrome c. These data suggest that megalin may play a significant role as a renal reabsorption receptor for the uptake of insulin and other low molecular weight polypeptides from the glomerular filtrate.

Megalin/gp330 is an endocytic receptor that internalizes multiple ligands including apolipoproteins E (apo E) and B100 (apo B). Megalin is the main antigenic target in passive Heymann nephritis (pHN), where it binds circulating autoantibodies leading to the formation of subepithelial immune deposits (ID)-the hallmark of pHN. Apo E and apo B were found recently to accumulate within these IDs, and evidence was provided that their lipids may undergo peroxidation, causing glomerular basement membrane damage and proteinuria. Here we investigated if ID-forming antimegalin IgG can inhibit the binding and internalization of apo E-betaVLDL (very low density lipoprotein) by megalin, and lead to their accumulation within IDs. By immunoelectron microscopy, apo E and apo B were detected in clathrin-coated pits and multivesicular bodies of podocytes in control rats, suggesting that the uptake of lipoproteins is a constitutive function of the glomerular epithelium. When pHN was induced by intravenous injection of antimegalin IgG, apo E and apo B were found within IDs by immunofluorescence and immunoelectron microscopy. Bound antibodies eluted from glomeruli of rats with pHN were found to inhibit the binding and internalization of apo E-enriched betaVLDL by megalin. These results indicate that pHN-inducing antimegalin IgG is capable of interfering with the uptake of lipoproteins by megalin in vivo during the formation of IDs.

Megalin (gp330) is a member of the low-density lipoprotein receptor gene family. Like other members of the family, it is an endocytic receptor that binds a number of specific ligands. Megalin also binds the receptor-associated protein (RAP) that serves as an exocytic traffic chaperone and inhibits ligand binding to the receptor. To investigate the fate of megalin/RAP complexes, we bound RAP glutathione-S-transferase fusion protein (RAP-GST) to megalin at the surface of L2 yolk sac carcinoma cells and followed the trafficking of the complexes by immunofluorescence and immunogold labeling and by their distribution on Percoll gradients. We show that megalin/RAP-GST complexes, which are internalized via clathrin-coated pits, are delivered to early endosomes where they accumulate during an 18 degrees C temperature block and colocalize with transferrin and transferrin receptor. Upon release from the temperature block, the complexes travel to late endosomes where they colocalize with rab7 and can be coprecipitated with anti-RAP-GST antibodies. Dissociation of the complex occurs in late endosomes and is most likely triggered by the low pH (approximately 5.5) of this compartment. RAP is then rapidly delivered to lysosomes and degraded whereas megalin is recycled to the cell surface. When the ligand, lipoprotein lipase, was bound to megalin, the receptor was found to recycle through early endosomes. We conclude that in contrast to receptor/ligand complexes, megalin/RAP complexes traffic through late endosomes, which is a novelty for members of the low-density lipoprotein receptor gene family.

Megalin (gp330), an epithelial endocytic receptor, is a major target antigen of Heymann nephritis (HN), an autoimmune disease in rats. To elucidate the mechanisms of HN, we have mapped a pathogenic epitope in megalin that binds anti-megalin antibodies. We focused our attention on four clusters of cysteine-rich, low density lipoprotein receptor (LDLR) ligand binding repeats in the extracellular domain of megalin because they represent putative ligand binding regions and therefore would be expected to be exposed in vivo and to be able to bind circulating antibodies. Rat megalin cDNA fragments I through IV encoding the first through fourth clusters of ligand-binding repeats, respectively, were expressed in a baculovirus system. All four expression products were detected by immunoblotting with two antisera capable of inducing passive HN (pHN). When antibodies eluted from glomeruli of rats with pHN were used for immunoblotting, only the expression product encoded by fragment II was detected. This indicates that the second cluster of LDLR ligand binding repeats is directly involved in binding anti-megalin antibodies and in the induction of pHN. To narrow the major epitope in this domain, fragment II was used to prepare proteins sequentially truncated from the C- and N-terminal ends by in vitro translation. Analysis of the truncated translation products by immunoprecipitation with anti-megalin IgG revealed that the fifth ligand-binding repeat (amino acids 1160-1205) contains the major epitope recognized. This suggests that a 46-amino acid sequence in the second cluster of LDLR ligand binding repeats contains a major pathogenic epitope that plays a key role in pHN. Identification of this epitope will facilitate studies on the pathogenesis of HN.

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 receptor-associated protein, RAP, is a chaperonin-like molecule that binds to two members of the low density lipoprotein receptor (LDLR) superfamily-megalin (gp330) and the LDL receptor-related protein (LRP). In F9 embryonal carcinoma cells, expression of RAP mRNA increases when differentiation is induced with retinoic acid and dibutyryl-cyclic AMP. We have investigated the expression of megalin and LRP and their interaction with RAP in F9 cells using biochemical and immunocytochemical methods. Both receptors are expressed in uninduced F9 cells, but only megalin co-precipitates with RAP. When F9 cells were induced to differentiate into parietal endoderm, the expression of megalin was dramatically increased. The expression of megalin exceeded that of LRP and RAP by an order of magnitude and both receptors co-precipitated with RAP. By immunoelectron microscopy, megalin and LRP were localized to clathrin-coated pits at the cell surface in both undifferentiated and differentiated F9 cells, whereas RAP was found mainly in the ER. A sizeable pool of LRP was also detected in the ER. When F9 cells were grown in suspension in the presence of RA and induced to develop into embryoid bodies, the expression of megalin and LRP segregated into different cell types: megalin was found in the outer epithelial layer and LRP in the stem cells of the inner core. Our results demonstrate that F9 cells induced to differentiate in monolayer culture express megalin, LRP and RAP, and RAP is capable of interacting simultaneously with both receptors. In embryoid bodies the expression of megalin and LRP diverges, and only megalin is expressed in the outer epithelial layer.(ABSTRACT TRUNCATED AT 250 WORDS)

The receptor-associated protein (RAP) specifically associates with gp330 and the low density lipoprotein (LDL) receptor-related protein (LRP), the two newest members of the LDL receptor gene family. Results obtained by ligand blotting, affinity chromatography, and density-gradient sedimentation demonstrate that RAP binds to both receptors with high affinity and that the binding is Ca2+ dependent. RAP also binds heparin and is identical to a mouse heparin binding protein (HBP-44) identified in a teratocarcinoma cell line (F9). While biochemical studies have shown that RAP is present on the cell surface and is an effective inhibitor of ligand binding to gp330 and LRP, immunocytochemical findings indicate that RAP is most abundant in the endoplasmic reticulum lumen and may function in receptor folding and/or trafficking. To facilitate the characterization of RAP's function(s) we have mapped its gp330 and heparin binding sites by performing direct binding studies on fusion proteins representing overlapping domains of RAP. gp330 was found to bind to two separate sites on RAP--i.e., between amino acids 85-148 and 178-248. Binding studies with radiolabeled heparin indicate that the heparin binding site is between amino acids 261 and 323, which is consistent with our previously proposed site (residues 287-306) based on the amphipathic nature of the C terminus of RAP. These data demonstrate that the gp330 and heparin binding sites and the Heymann nephritis pathogenic epitope (amino acids 1-86) demonstrated earlier are represented by distinct domains of the RAP polypeptide.

Heymann nephritis is an experimental autoimmune disease model for human membranous nephropathy. We have recently identified a pathogenic epitope, clone 14 (C14), responsible for formation and deposition of glomerular immune complexes that is contained within the small subunit of the Heymann nephritis antigenic complex (HNAC). HNAC is a heterodimer composed of a large subunit designated gp330 and a smaller (44 kDa) subunit, which is immunologically identical to the receptor-associated protein. In this study, we prepared antibodies to fusion proteins with C-terminal deletions in the C14 sequence and assessed their ability to promote formation of immune deposits (IDs). When IgG specific for the shortest truncated fusion protein (C14/delta 3; 86 amino acids) was injected into rats, small IDs developed. In contrast, when IgG raised against the full-length C14 sequence was depleted of its reactivity toward the C14/delta 3 fusion protein (C14/delta 3-fp), no IDs could be detected. These data indicate that at least one pathogenic epitope is contained within the N-terminal 86 amino acids of C14. Since the IDs induced with the C14/delta 3-fp-specific IgG are smaller than those induced with the poly-epitope-specific anti-gp330 antibodies, it is likely that other epitopes in addition to those expressed by the C14/delta 3-fp are required for formation and growth of immune complexes.

Background A major portion of available fatty acids for adipocyte uptake is derived from lipoprotein lipase (LPL)-mediated hydrolysis of circulating lipoprotein particles. In vivo studies aimed at identifying the precise role of adipocyte-derived LPL in fat storage function of adipose tissue have been unable to provide conclusive evidence due to compensatory mechanisms that activate endogenous fatty acid synthesis. To address this gap in knowledge, we have measured the effect of reducing adipocyte LPL expression on intracellular lipid accumulation using a well-established cultured model of adipocyte differentiation. Methods siRNA specific for mouse LPL was transfected into 3T3-L1 adipocytes. Expression of LPL was measured by quantitative real-time PCR and cell surface-associated LPL enzymatic activity was measured by colorimetric detection following substrate (p-nitrophenyl butyrate) hydrolysis. Apolipoprotein CII and CIII expression ratios were also measured by qRT-PCR. Intracellular lipid accumulation was quantified by Nile Red staining. Results During differentiation of 3T3-L1 pre-adipocytes, LPL mRNA expression increases 6-fold resulting in a 2-fold increase in cell surface-associated LPL enzymatic activity. Parallel to this increase in LPL expression, we found that intracellular lipids increased ~10-fold demonstrating a direct correlation between adipocyte-derived LPL expression and lipid storage. We next reduced LPL expression in adipocytes using siRNA transfections to directly quantify the contributions of adipocyte-derived LPL to lipid storage, This treatment reduced LPL mRNA expression and cell surface-associated LPL enzymatic activity to ~50% of non-treated controls while intracellular lipid levels were reduced by 80%. Exogenous addition of purified LPL (to restore extracellular lipolytic activity) or palmitate (as a source of free fatty acids) to siRNA-treated cells restored intracellular lipid levels to those measured for non-treated controls. We also found that adipocytes express apolipoprotein CII and CIII and, in addition, the apoCII/apoCIII ratio remains largely unchanged in cells with reduced LPL expression. Conclusion We provide evidence that adipocyte-derived LPL is required for efficient fatty acid uptake and storage, and that adipocytes express their own source of apoCII and apoCIII for regulating extracellular LPL activity. These findings demonstrate that adipocytes are capable of producing the necessary enzymatic components and co-factors for efficient lipid storage independent of vascular sources.

Background Transport of fatty acids within the cytosol of adipocytes and their subsequent assimilation into lipid droplets has been thoroughly investigated; however, the mechanism by which fatty acids are transported across the plasma membrane from the extracellular environment remains unclear. Since triacylglycerol-rich lipoproteins represent an abundant source of fatty acids for adipocyte utilization, we have investigated the expression levels of cell surface lipoprotein receptors and their functional contributions toward intracellular lipid accumulation; these include very low density lipoprotein receptor (VLDL-R), low density lipoprotein receptor-related protein (LRP), and heparan sulfate proteoglycans (HSPG).

Total protein content of P-815 mastocytoma cells decreases and then increases in response to initiation of a new growth cycle. As the level increases, the rate of synthesis declines. Both events occur prior to any decrease in the rate of cellular proliferation. These temporal relationships indicate that the rate of protein synthesis reflects the intracellular concentration of protein rather than the cellular growth rate, as has been hypothesized. Pyruvate kinase protein metabolism differs from that of total protein in three ways: (a) accumulation does not stop, (b) the rate of synthesis does not decrease, and (c) only the rate of pyruvate kinase degradation is altered by a factor present in conditioned media. These observations suggest that there are specific mechanisms regulating pyruvate kinase at a post-transcriptional level.

The seafloor of the central part of lower Cook Inlet, Alaska, is characterized by the presence of different sizes and types of bedforms. The bedforms in the sandy sediments include straight-crested to sinuous to lunate ripples, small, medium, and large sand waves, sand ridges, sand ribbons, and sand patches. In addition, rocky and pebbly seafloor has been identified. The water depth ranges from 25 to 120 m, and surface currents average 3.8 kt (2 m/s). Bottom currents have been measured at as much as 42 cm/s at 1 m above bottom. Underwater television observations have shown that the rate of sand transport is lower than expected because small amounts of clay and organic matter appear to inhibit remobilization. Only during the last 1 to 2 h of ebb and flood stages of spring tides, and during storms, does significant transport occur. Comparison of data from high-resolution seismic profiling systems, side-scan sonar, bottom television and camera, and bottom sampling shows that bottom and bedform interpretations based solely on sonographs can be in error. Measuring the length of ‘acoustic shadows’ on sonographs to obtain bedform heights gives dimensions that are too large by factors of 3–7. Bottom television investigations revealed that the troughs between small sand waves are flat and carpeted by shell fragments. Such coarse material has a high acoustic reflectance that is not related to slope or height and can lead to false interpretations on bedform dimensions. Our observations have shown that small sand waves commonly superimposed on larger ones are slightly higher than those present on flat hard bottom but are still less than calculated from acoustic shadows. Where the bottom is rather smooth or contains elevations small enough to be masked by bathymetric ‘noise’ caused by the pitching of the vessel, sonographs typically show either small sand waves, sand ribbons, sand patches, rocks, or smooth bottom. The smooth-bottom category can vary widely from ripples to gravelly or shelly or to small rocks with biological overgrowth as verified by television observations. Our observations have clearly demonstrated the need for an integrated multi-scale observation and sampling program in order to classify the bottom characteristics and to provide quantitative data for transport calculations.

Normal and abnormal breast samples of women were analyzed for multiple molecular forms of lactate dehydrogenase and glucose 6-phosphate dehydrogenase, using acrylamide disc gel electrophoresis and specific enzyme staining techniques for separation and quantitation. Infiltrating ductal carcinomas demonstrated a significant increase (three to six-fold) in the proportion of LDH-5 compared to samples of normal breast, fibrocystic disease and fibroadenoma, indicative of a shift toward the muscle-type lactate dehydrogenase in neoplasia. For glucose 6-phosphate dehydrogenase, carcinomas were found to contain increased proportions of the fastest migrating species, G6PD-I. Total enzyme activity/mg DNA was elevated in meoplastic tissues. Little or no alteration in isoenzyme profiles could be related to menopausal status of the patient.

P-815 mastocytoma cells increase the level of pyruvate kinase (PK) expression in response to chloroform-methanol extracts of conditioned media, butyrate, and dibutyryl cyclic AMP (but2cAMP) plus theophylline. The butyrate effect is indomethacin sensitive, suggesting a prostaglandin (PG) is the active signaling factor. Moreover, the chloroform-methanol extracts contain PGE2 and PGF2 alpha and additions of the latter enhance PK activity. PGE2 alone has little or no effect but acts synergistically with PGF2 alpha. These data show that PGF2 alpha can regulate PK levels. On the other hand, other factors may also be active, since the endogeneous and the but2cAMP plus theophylline effects are indomethacin insensitive. Most of the factors that increase PK activity also inhibit cellular growth; however, regulation of PK expression can be uncoupled from growth inhibition.

Lower Cook Inlet in Alaska has high‐ tidal currents that average 3–4 knots and normally reach a peak of 6–8 knots. The bottom has an average depth of about 60–70 m in the central part of the inlet that deepens toward the south. Several types of bedforms, such as sand waves, dunes, ripples, sand ribbons, and lag deposits form a microtopography on the otherwise smooth seafloor. Each bedform type covers a small field, normally a few hundred to a few thousand meters wide, and usually several kilometers long parallel to the tidal flow. High‐resolution seismic systems, side‐scan sonar and bottom television were used to study these bedforms. Large sand waves with wavelengths over 300 m and wave heights up to 10 m were observed. Fields of ebb‐oriented or flood‐oriented asymmetric bedforms commonly grade into more symmetric shapes. Several orders of smaller sand waves and dunes cover the flanks of the very large bedforms. The crest directions of both size groups are normally parallel, but deviations of up...

The specific activities of pyruvate kinase and phosphofructokinase but not lactate dehydrogenase increase as P-815 mastocytoma cells approach the stationary phase. During this growth period, the rates of uptake of labelled precursors into DNA, RNA and total protein decreases. On the other hand, the pyruvate kinase protein level changes in parallel with activity. Although the K-isozyme is the primary form of pyruvate kinase expressed, some M-type subunit is also present and both forms undergo an increase in specific activity. In addition, pyruvate kinase expression is also elevated by adding cAMP analogues with theophylline, butyrate or conditioned media. This increased level of expression is hypothesized to be a secondary event associated with a differentiation-like-induced expression of the mast cell phenotype.

SummaryHuman infiltrating ductal carcinoma of the breast, fibrocystic disease of the breast, and normal human mammary tissues were assayed for their concentration of nucleic acid, lipids, and 11 selected enzymes involved in carbohydrate, lipid, and amino acid metabolism. Comparing infiltrating ductal carcinomas to normal mammary tissue, striking elevations (p

ABSTRACT The central part of lower Cook Inlet, Alaska, is covered by marine bedforms of varying size and type. Swift tidal currents sweep the area, and assessment of the effects of these currents on the sandy bottom materials is essential to the industrial development of lower Cook Inlet. As part of the summer 1978 field program, we selected a small area for detailed observations. In addition to high resolution seismic profiling and side-scan sonar, we conducted bottom television/camera observations and vertical current-velocity profiling. Time-series data on bottom currents, bottom pressure, near-bottom suspended matter, and bottom photography were obtained with the Geological Processes Bottom Environmental (GElJPROBE) system during August and September 1978 in the study area. An anchor station was occupied for 26 hours over 9-m-high sand waves after the site was surveyed in detail. While the vessel was anchored, current-meter profiles and bottom television/camera observations were made. In addition suspended sediment samples were obtained during deployment of the GEOPROBEs. Surface currents showed a rotary tidal-current pattern with peak velocities of about 100 cm/s. Near the bottom we observed a trimodal current-direction distribution and measured unexpectedly low velocities that were possibly due to obstruction by the bedforms. We observed sand transport along the bottom only during the last hour of both the ebb-tidal and flood-tidal cycles of the spring tide, when velocities exceeded 30 cm/s at 1 m above bottom. The complex bathymetry of the large sand waves, characterized by rapid variations in the elevation of crests, complicates comparison of the anchor station results. Short-term deployments of GEOPROBEs in July 1978 showed that most of the transport of bottom material, with a mean grain size of fine to medium sand, occurs at threshold current velocities of at least 20 cm/s at 1 m above bottom. The highest transport rate in the study area occurred during ebb tide near the crest of a large sand wave. GEOPROBE bottom protographs show that ripples near the crests of large sand waves reverse orientation from ebb to the next flood. Suspended-sediment samples and light-beam transmission data also show active resuspension during peak tidal currents in both the crest and trough area. During neap tides, however, a negligible amount of bottom sediment moves in the sand-wave trough, despite bottom current velocities of more than 20 cm/so an admixture of silt and organic detritus may inhibit sediment response in the troughs. INTRODUCTION Cook Inlet is a large estuary in south-central Alaska that extends from Anchorage to the Pacific Ocean. Tidally dominated currents control the hydrographic regime and may have a significant influence on surficial bottom sediments. Response of the sediments to strong tidal currents was part of the environmental geologic studies conducted by the U.S. Geological Survey during 1976-78 on board the research vessel Sea Sounder. Surveys were conducted utilizing high-resolution seismic reflection profiling and side-scan sonar. Several stations were occupied for bottom sampling, underwater television and camera observations, vertical current-velocity profiles, and deployment of GEOPROBEs (Geological Processes Bottom Environmental system; Cacchione and Drake, 1979).

ABSTRACT Geologic environmental studies in lower Cook Inlet Alaska, indicate that in addition to earthquake accelerations, nuees ardentes from volcanic eruptions, and shallow gas, the only other significant potential geologic hazard is that associated with sand motion and migration of bed forms found in the central part of this area. Four major size groups of bed forms have been observed:small. asymmetric ripples normally less than 30 cm long with wave heights of about 5 to 7 cm,small sand waves with wave lengths less than a few tens of meters and wave heights less than a few meters,medium-size sand waves and small dunes with wave lengths ranging from 50 to 150 m and wave heights less than 5 m, andlarge sand waves, dunes, and sand ridges with wave lengths ranging from 350 to 1000 m and wave heights from 5 to 10 m. Several size classes typically are superimposed on each other. Sand grains either rollover the bottom or form sheet flows, the latter moving as fast as 30 cm/s over the crests of the larger bed forms. High-resolution seismic profiling and side-scan sonar with accurate Mini-Ranger positioning were used to map two areas in detail. Underwater television, bottom photography, and surficial sediment sampling gave additional information. Maintaining station position is often difficult due to the high tidal currents with peak velocities ranging from 3 to 5 nots. Scanning electron microscopic examinations of surface textures of quartz grains imply that there is no substantive net transport of grains. Drill cuttings, released during a period of 3 months, were incorporated to a depth of at least l3.5 cm. However, concentrations at 5.5 and 13 cm depth suggest transport by small ripples over small sand waves. Although more transport may occur during heavy runoff and storm periods, the present data-indicate substantial transport of sand over large bed forms at most times and possible additional seasonal mass movement over smaller ones. Understanding stability and migration of bed forms is a high priority for scientific and industrial interests. INTRODUCTION Cook Inlet is a large tidal inlet located in south central Alaska (Fig. 1), extending from Anchorage to the Pacific Ocean. Tidal currents are the main agents of mass water movement up and down the inlet. The influence of the currents on the bottom was one subject of an environmental geology study conducted during the summers of 1976 and 1977 on board the U.S. Geological Survey R/V SEA SOUNDER. High-resolution seismic profiling, side-scan sonar surveys, surficial sediment sampling, underwater television, and bottom photographs were made in lower Cook Inlet (Bouma and Hampton, 1976; Hampton and Bouma, 1976; Bouma and others, 1977a, 1978). The central part of lower Cook Inlet is characterized by a variety of bed form types. Our studies concentrated on movements of unconsolidated noncohesive grains as individual particles and on the influence such motion has on migration of bedforms. Two smaller areas were selected for detailed studies, one near the D/V OCEAN RANGER, and the other off Anchor Point (Fig. 2).

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...

Background Adipocytes express inflammatory mediators that contribute to the low-level, chronic inflammation found in obese subjects and have been linked to the onset of cardiovascular disorders and insulin resistance associated with type 2 diabetes mellitus. A reduction in inflammatory gene expression in adipocytes would be expected to reverse this low-level, inflammatory state and improve cardiovascular function and insulin sensitivity. The natural products, curcumin and resveratrol, are established anti-inflammatory compounds that mediate their effects by inhibiting activation of NF-κB signaling. In the present study, we examined if these natural products can inhibit NF-κB activation in adipocytes and in doing so reduce cytokine expression. Methods Cytokine (TNF-α, IL-1β, IL-6) and COX-2 gene expression in 3T3-L1-derived adipocytes was measured by quantitative real-time PCR (qRT-PCR) with or without TNFα-stimulation. Cytokine protein and prostaglandin E2 (PGE2) expression were measured by ELISA. Effects of curcumin and resveratrol were evaluated by treating TNFα-stimulated adipocytes with each compound and 1) assessing the activation state of the NF-κB signaling pathway and 2) measuring inflammatory gene expression by qRT-PCR and ELISA. Results Both preadipocytes and differentiated adipocytes express the genes for TNF-α, IL-6, and COX-2, key mediators of the inflammatory response. Preadipocytes were also found to express IL-1β; however, IL-1β expression was absent in differentiated adipocytes. TNF-α treatment activated NF-κB signaling in differentiated adipocytes by inducing IκB degradation and NF-κB translocation to the nucleus, and as a result increased IL-6 (6-fold) and COX-2 (2.5-fold) mRNA levels. TNF-α also activated IL-1β gene expression in differentiated adipocytes, but had no effect on endogenous TNF-α mRNA levels. No detectable TNFα or IL-1β was secreted by adipocytes. Curcumin and resveratrol treatment inhibited NF-κB activation and resulted in a reduction of TNF-α, IL-1β, IL-6, and COX-2 gene expression (IC50 = 2 μM) and a reduction of secreted IL-6 and PGE2 (IC50 ~ 20 μM). Conclusion Curcumin and resveratrol are able to inhibit TNFα-activated NF-κB signaling in adipocytes and as a result significantly reduce cytokine expression. These data suggest that curcumin and resveratrol may provide a novel and safe approach to reduce or inhibit the chronic inflammatory properties of adipose tissue.