On the bubble-bubbleless ocean continuum and its meaning for the LiDAR equation: LiDAR measurement of underwater bubble properties during storm conditions.

This paper presents the NRL shipboard LiDAR and the first LiDAR dataset of underwater bubbles. The meaning of these LiDAR observations, the algorithms used and their current limitations are discussed. The derivation of the LiDAR multiple scattering regime is derived from the LiDAR observations and theory. The detection of the underwater bubble presence and their depth is straightforward to estimate from the depolarized laser return. This dataset strongly suggest that the whitecaps term in the LiDAR equation formalism needs to be revisited. The retrieval of the fraction of air volume within a given volume of water (void fraction) is possible and the algorithm is stable with a simple ocean backscatter LiDAR system. The accuracy of the void fraction retrieval will increase significantly with future developments.

Classical and alternative NF-κB signaling cooperate in regulating adipocyte differentiation and function.

BACKGROUND AND OBJECTIVE: Inflammation of adipose tissue (AT) is a central mediator of insulin resistance. However, the molecular mechanisms triggered by inflammatory cells are not fully understood. The aim of this study was to analyze the metabolic functions of lymphotoxin-ß-receptor (LTßR)-mediated alternative NF-κB signaling in adipocytes and to reveal its effects on body weight and insulin sensitivity in vivo. METHODS: RelB(FatKO) mice and littermate controls were treated with LTßR agonistic antibody (α-LTßR) or a LTßR antagonist (LTßR:Ig fusion protein) after feeding a high-fat diet or standard diet. Mice were analyzed by insulin tolerance and glucose tolerance tests prior to analysis by necropsy and qRT-PCR of abdominal white adipose tissue. 3T3-L1 preadipocytes and mouse embryonic fibroblasts were used for differentiation and expression analysis after treatment with α-LTßR and differentiation to adipocytes. The molecular mechanism was elucidated by chromatin immunoprecipitation and combinatorial treatment with α-LTßR and tumor necrosis factor (TNF). RESULTS: RelB(FatKO) mice showed improved insulin sensitivity despite increased adiposity and adipocyte hypertrophy. LTßR-induced activation of p52-RelB in 3T3-L1 cells attenuated adipogenesis and modulated adipocyte functions via transcriptional downregulation of peroxisome proliferator-activated receptor γ (PPARγ). This LTßR-mediated pathway was synergistically regulated via a TNF-induced increase in p100 and RelB expression and nuclear translocation. CONCLUSIONS: Our data describe an anti-adipogenic action of LTßR signaling and a novel synergism of alternative and classical NF-κB signaling in the regulation of adipocytes. In conclusion, this strong synergism between the two NF-κB pathways shows a method to inhibit adipocyte differentiation and to improve insulin sensitivity and can be a potential target to treat metabolic disorders more efficiently than with other known drugs.

Observation of polarized positrons from an undulator-based source.

An experiment (E166) at the Stanford Linear Accelerator Center has demonstrated a scheme in which a multi-GeV electron beam passed through a helical undulator to generate multi-MeV, circularly polarized photons which were then converted in a thin target to produce positrons (and electrons) with longitudinal polarization above 80% at 6 MeV. The results are in agreement with GEANT4 simulations that include the dominant polarization-dependent interactions of electrons, positrons, and photons in matter.

Biology of HIF-1alpha.

The increase in body size of humans and other vertebrates requires a physiological infrastructure to provide adequate delivery of oxygen to tissues and cells to maintain oxygen homeostasis. The heart, lungs and the vasculature are all part of a highly regulated system that ensures the distribution of the precise amount of oxygen needed throughout the mammalian organism. Given its fundamental impact on physiology and pathology, it is no surprise that the response of cells to a lack of oxygen, termed hypoxia, has been the focus of many research groups worldwide for many decades now. The transcriptional complex hypoxia-inducible factor has emerged as a key regulator of the molecular hypoxic response, mediating a wide range of physiological and cellular mechanisms necessary to adapt to reduced oxygen.

Observation of plasma focusing of a 28.5 GeV positron beam.

The observation of plasma focusing of a 28.5 GeV positron beam is reported. The plasma was formed by ionizing a nitrogen jet only 3 mm thick. Simultaneous focusing in both transverse dimensions was observed with effective focusing strengths of order tesla per micron. The minimum area of the beam spot was reduced by a factor of 2.0+/-0.3 by the plasma. The longitudinal beam envelope was measured and compared with numerical calculations.

Intracellular apolipoprotein E affects Amyloid Precursor Protein processing and amyloid Abeta production in COS-1 cells.

The apoE gene has been identified as a major susceptibility locus for late-onset Alzheimer's disease (LOAD). The epsilon4 allele greatly reduces age of onset of LOAD as compared to the wild-type 3 allele. The molecular mechanism(s) underlying the association has not yet been fully elucidated. The apoE protein has been shown to physically interact with the Abeta region of the Amyloid Precursor Protein (APP), but also with the ectodomain of the APP holoprotein itself. In this study we have used apoE fusion proteins containing either the ER retention sequence KDEL or trans-Golgi network (TGN) signal sequence in order to define potential apoE-mediated alterations in APP protein processing. Co-expression and pulse-chase experiments showed that a functional apoE:APP interaction occurs intracellularly which directly affects maturation and subsequently the secretion kinetics of APP. In addition, an epsilon4 allele-specific induction of Abeta production has been demonstrated. apoE3 resulted in increased Abeta production only when targeted to the ER, as observed in cells transfected with an apoE3KDEL fusion protein as well as following treatment with brefeldin A. The findings suggest that in cells that express both apoE and APP, such as astrocytes and microglia, a functional apoE:APP interaction may occur which modulates APP processing and Abeta production.

Aspartyl protease inhibitor pepstatin binds to the presenilins of Alzheimer's disease.

Mutations in the presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease by altering gamma-secretase cleavage of the amyloid precursor protein, the last step in the generation of Abeta peptide. Ablation of presenilin (PS) genes, or mutation of two critical aspartates, abolishes gamma-secretase cleavage, suggesting that PS may be the gamma-secretases. Independently, inhibition experiments indicate that gamma-secretase is an aspartyl protease. To characterize the putative gamma-secretase activity associated with presenilins, lysates from human neuroblastoma SH-SY5Y and human brain homogenates were incubated with biotin derivatives of pepstatin, followed by immunoprecipitation of PS and associated proteins, and biotin detection by Western blotting. Precipitation with PS1 antibodies, directed to either N-terminal or loop regions, yielded the same 43 kDa band, of apparent molecular mass consistent with that of full-length PS1, although it may represent an aspartyl protease complexed with PS1. Incubation of cell lysates with pepstatin-biotin, followed by streptavidin precipitation and PS1 Western blotting, revealed PS1 fragments and full-length protein, indicating that pepstatin-biotin bound to both cleaved and uncleaved PS1. Binding could be competed by gamma-secretase inhibitor L-685,458 and could not be achieved with a PS1 mutant lacking the two transmembrane aspartates. Pepstatin-biotin was also shown to bind to PS2. PS1 was specifically absorbed to pepstatin-agarose, with an optimal pH of 6. Binding of pepstatin-biotin to PS1 from lymphocytes of a heterozygous carrier of pathologic exon 9 deletion was markedly decreased as compared to control lymphocytes, suggesting that this PS1 mutation altered the pepstatin binding site.

Presenilin 2 expression in neuronal cells: induction during differentiation of embryonic carcinoma cells.

Mutations in the presenilin 1 and 2 (PS1 and PS2) genes cause most cases of early onset Alzheimer's disease. The genes encode two homologous multipass membrane proteins. Since the endogenous expression of PS2 has been poorly analyzed to date, we studied PS2 expression and localization in cultured human neuroblastoma cells and mouse neuronal cells. PS2 was mainly detected as a full-length protein of about 52 kDa in these cells and in brain, in contrast to PS1 that is mainly detected as endoproteolytic N-terminal and C-terminal fragments. Using immunofluorescence we found that like PS1, PS2 colocalized with markers of the endoplasmic reticulum-Golgi intermediate compartment, ERGIC-53 and beta-COP. Double labeling for PS1 and PS2 indicated that both proteins are colocalized in neuroblastoma SH-SY5Y cells. To study PS2 expression during differentiation, mouse embryonic carcinoma P19 cells were treated with retinoic acid. We found minimal PS2 expression in undifferentiated cells, an increase from day 2, and a maximum at day 8 after treatment. PS1 expression remained constant during this period. The differential expression of PS1 and PS2 within the P19 cells following retinoic acid treatment indicates different utilization or temporal requirements for these proteins during neuronal differentiation.

Presenilin I expression in yeast lowers secretion of the amyloid precursor protein.

Presenilin (PS) mutations are associated with early-onset Alzheimer's disease and PS proteins are involved with gamma-secretase cleavage of the amyloid precursor protein, APP. We have shown previously that alpha-, beta- and gamma-secretase cleavages of APP are conserved in Pichia pastoris. Here, we report co-expression of APP and PSI in P. pastoris and show by immunoelectron microscopy colocalization of these two proteins in expanded endoplasmic reticulum. Western blot analysis indicates a drastic reduction of both alpha- and beta-secretase products. A relative increase in beta-secretase product derived from immature APP is also observed, pointing to a beta-secretase activity of P. pastoris associated with the early secretory pathway.

Interaction of the Presenilins with the Amyloid Precursor Protein (APP).

The genes encoding presenilin-1 (PS1) and presenilin-2 (PS2) were identified as the genes that harbour mutations that cause more than 60% of early onset familial Alzheimer's disease cases (FAD) (1-3). So far, more than 40 missense mutations have been described for presenilin-1 and two have been found in the gene coding for presenilin-2 (reviewed in refs. 4 and 5). Carriers of mutated presenilin genes develop in their brain neuropathological changes characteristic of Alzheimer's disease including the deposition of amyloid Aß peptide. The latter is released from its cognate amyloid precursor protein (APP) by a two-step proteolytic conversion: first, proteolysis of APP by ß-secretase, which releases the N-terminus of Aß, and second, conversion of the remaining fragment by γ-secretase, which cleaves within the predicted transmembrane region of APP. This releases the C-terminus of Aß, which may end either at position 40 or, to a lesser extent, at position 42 (reviewed in ref. 6). The latter species, Aß(1-42), is more prone to aggregation and deposition than Aß(1-40) and is produced at higher levels in the brains and primary fibroblasts of FAD patients carrying PS missense mutations (7). The same result was obtained when cultured cells transfected with mutated PS1 orPS2, or transgenic mice harboring missense PS1 were analyzed for the production of Aß(1-42): in every case increased amounts of the longer Aß(1-42) species were observed (8-10). The mechanisms by which mutations in the PS genes affect the proteolytic processing of APP by γ-secretase have not been resolved in detail. There are two possibilities by which the normal processing of APP may be disturbed: either mutations in the presenilins affect APP metabolism in an indirect way by modulation of proteases or interaction with proteins involved in APP intracellular routing, or presenilins may modulate APP processing directly through physical interactions with APP. Such a direct interaction between presenilins and APP was first demonstrated by us for PS2 (11). Later on, formation of stable complexes with APP was reported not only for PS2 but also for PS1 (12,13,13a).

Proteolytic processing of the Alzheimer's disease amyloid precursor protein within its cytoplasmic domain by caspase-like proteases.

Alzheimer's disease is characterized by neurodegeneration and deposition of betaA4, a peptide that is proteolytically released from the amyloid precursor protein (APP). Missense mutations in the genes coding for APP and for the polytopic membrane proteins presenilin (PS) 1 and PS2 have been linked to familial forms of early-onset Alzheimer's disease. Overexpression of presenilins, especially that of PS2, induces increased susceptibility for apoptosis that is even more pronounced in cells expressing presenilin mutants. Additionally, presenilins themselves are targets for activated caspases in apoptotic cells. When we analyzed APP in COS-7 cells overexpressing PS2, we observed proteolytic processing close to the APP carboxyl terminus. Proteolytic conversion was increased in the presence of PS2-I, which encodes one of the known PS2 pathogenic mutations. The same proteolytic processing occurred in cells treated with chemical inducers of apoptosis, suggesting a participation of activated caspases in the carboxyl-terminal truncation of APP. This was confirmed by showing that specific caspase inhibitors blocked the apoptotic conversion of APP. Sequence analysis of the APP cytosolic domain revealed a consensus motif for group III caspases ((IVL)ExD). Mutation of the corresponding Asp664 residue abolished cleavage, thereby identifying APP as a target molecule for caspase-like proteases in the pathways of programmed cellular death.

Screening the human bradykinin B2 receptor gene in patients with cardiovascular diseases: identification of a functional mutation in the promoter and a new coding variant (T21M).

To elucidate if genetic variants in the bradykinin B2 receptor (B2) gene occur that could affect receptor expression and function, we screened for mutations in the promoter and in the coding region of the human B2 gene. In our initial study we analyzed 92 consecutive, unrelated subjects (including 25 patients with hypertrophic cardiomyopathy, 18 patients with dilated cardiomyopathy (DCM), 25 patients with hypertension, 18 patients with coronary heart disease, and 6 patients with valvular heart disease) using nonradioactive polymerase chain reaction-single-strand conformation polymorphism analysis as mutation screening method. We detected eight as yet unknown polymorphic sites in the promoter region of the B2 gene (-845 C/T, -704 C/T, -649 insG, -640 T/C, -536 C/T, -412 C/G, -143 C/T and -78 C/T) with allele frequencies between 0.5 and 13%. One of them (-412 C/G) destroys a Sp1 binding site and abolishes protein binding to this Sp1 site in human umbilical vein endothelial cells and human vascular smooth muscle cells. In the protein-coding region one new coding variant (T21M) with the potential to create a truncated receptor isoform was detected. We determined the frequency of the promoter variant at position -412 (C --> G) and the newly identified coding variant (T21M) in extended samples of 69 patients with HCM, 163 patients with DCM, 109 patients with hypertension, and 173 healthy anonymous blood donors. The promoter variant (-412 C/G) was found in one blood donor and the T21M mutation was not found in the control population. Therefore, it appears that these mutations are rare events and the determination of clinical significance will be a demanding task in the future.

Intracellular and secreted Alzheimer beta-amyloid species are generated by distinct mechanisms in cultured hippocampal neurons.

Cerebral plaques containing beta-amyloid (beta A4) represent an invariant pathological feature of Alzheimer disease (AD). beta A4 is proteolytically generated from its parent molecule, amyloid precursor protein (APP). In non-neuronal cells beta A4 has been shown to be secreted via a pH-sensitive and endocytosis-dependent pathway, and this process, when occurring in the brain, is considered to play an important role in AD. In neurons the mechanisms of beta A4 production are not known. Here we have analyzed these mechanisms by expressing human APP and its mutant versions in hippocampal neurons using the Semliki forest virus system. We show that these cells initially generate two pools of beta A4, an extracellular and an intracellular, and only the extracellular pool is produced via a pH-sensitive and endocytosis-dependent pathway. Thus, hippocampal neurons are able to utilize an alternate pathway to produce intracellular beta A4. We also show that a common feature of two types of APP mutations ("Swedish" and "London") implicated in early-onset AD is their increased production of C-terminally elongated beta A4 (beta 42), both intra- and extracellularly. Since neurons are the only cells that produce substantial levels of intracellular beta A4 and also the main victims in AD, these findings may provide an important link between beta A4 and neurodegeneration.

Formation of stable complexes between two Alzheimer's disease gene products: presenilin-2 and beta-amyloid precursor protein.

Mutations in the presenilin genes are associated with early onset familial Alzheimer's disease and lead to increased accumulation of beta A4 peptide, the proteolytic product of the amyloid precursor protein (APP). To test whether presenilins interfere with APP metabolism, presenilin-2 (PS2) was coexpressed with APP in mammalian cells. Analysis of PS2 immunoprecipitates revealed that a fraction of APP was associated with the PS2 immunocomplexes. This non-covalent association was specific for the APP family of proteins and restricted to immature forms, occurring probably during transit through the endoplasmic reticulum. Additionally, coexpression with PS2 resulted in a decrease of APP secretion, suggesting a direct participation of presenilins in the intracellular sorting, trafficking and processing of APP molecules.

Human amyloid precursor-like protein 1--cDNA cloning, ectopic expression in COS-7 cells and identification of soluble forms in the cerebrospinal fluid.

Amyloid precursor-like protein 1 (APLP1) represents an integral membrane type 1 protein of unknown function which was originally cloned from a mouse cDNA library on the basis of sequence similarity with the Alzheimer's amyloid precursor protein (APP). Here we report on the molecular cloning and expression of the human APLP1 (hAPLP1). hAPLP1 consists of 650 amino acids, displays 89% identity on the amino acid level to its mouse homologue and has a calculated molecular mass of 72 kDa. hAPLP1 synthesized in a cell-free system displays an apparent molecular mass of approximately 80 kDa in SDS-containing gels and becomes N-glycosylated when the in vitro translation is performed in the presence of microsomes. The hAPLP1 cDNA was also expressed ectopically in COS-7 cells and the protein expression was analyzed by immunoprecipitation and western blotting. We have demonstrated that hAPLP1 represents a novel glycoprotein which carries both N- and O-linked glycans. Moreover, hAPLP1 undergoes limited proteolysis which results in the secretion of the carboxy-terminal truncated molecule into the cells conditioned medium. Examination of cells transfected with hAPLP1 cDNA by confocal laser microscopy reveals an intense perinuclear and Golgi staining, a pattern resembling the subcellular distribution of APP. Using a novel hAPLP1-specific antiserum, we identified soluble hAPLP1 in the human cerebrospinal fluid, which suggests that secretion of hAPLP1 from brain cells also takes place in vivo.

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP.

Fractions from commercial collagenase preparations: use in enzymic isolation of the islets of Langerhans from porcine pancreas.

Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the libration of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.

Alzheimer's disease betaA4 protein release and amyloid precursor protein sorting are regulated by alternative splicing.

We show here that alternative splicing influences the polarized secretion of amyloid precursor protein (APP) as well as the release of its proteolytic 3-4-kDa fragments betaA4 and p3. In Madin-Darby canine kidney II cells stably transfected with various APP isoforms and APP mutants, APPsec was consistently secreted basolaterally. In contrast, Madin-Darby canine kidney II cells transfected with L-APP677, which occurs naturally by alternative splicing of exon 15, secreted this isoform both apically and basolaterally, while maintaining the basolateral sorting of endogenous APPsec. This suggests that the alternative splicing of APP exon 15 modulates the polarized sorting of secretory APP. The same alternative splicing event also decreased the production of betaA4 relative to p3. This is the first example of alternative splicing regulating polarized trafficking of a secretory protein.

Extracellular matrix influences the biogenesis of amyloid precursor protein in microglial cells.

During axotomy studies, we discovered that the beta A4-amyloid precursor protein (APP) participates in immune responses of the central nervous system. Since microglia constitute the main immune effector cell population of this response, we used the murine microglial cell line BV-2 to analyze immune response-related APP expression. We show that interaction of microglia with the extracellular environment, particularly components of the extracellular matrix, affects APP secretion as well as intracellular APP biogenesis and catabolism. Fibronectin enhanced APP secretion and decreased the level of cellular mature transmembrane APP, whereas laminin and collagen caused a decrease in secretion and an accumulation of cellular mature APP and APP fragments. Our results demonstrate that APP plays a fundamental role in the regulation of microglial mobility, i.e. migration, initial target recognition, and binding. The decrease in APP secretion and the concomitant increase in cellular mature APP were accompanied by an accumulation of C-terminal APP fragments. Enrichment of APP and APP fragments is assumedly based on inhibition of catabolic processes that is caused by a disorganization of the actin microfilament network. These observations provide evidence that microglia, which are closely associated with certain amyloid deposits in the brain of Alzheimer patients, can play a key role in initial events of amyloidogenesis by initiating accumulation of APP and also of amyloidogenic APP fragments in response to physiological changes upon brain injury.

Raman scattering in ocean optics: quantitative assessment of internal radiant emission.

Raman-scattering activity in clear ocean waters is further documented from Monte Carlo simulations and optical data that are collected in the Sargasso Sea. A method is proposed, based on the anomalous absorption coefficient for a nonconservative irradiance field, to assess the percentile composition of internal radiant emission for the irradiance field at any depth.