Transfer and effects of 1,2,3,5,7-pentachloronaphthalene in an experimental food chain.

Polychlorinated naphthalenes are environmentally relevant compounds that are measured in biota at concentrations in the μg/kg lipid range. Despite their widespread occurrence, literature data on the accumulation and effects of these compounds in aquatic ecosystems are sparsely available. The goal of this study was to gain insights into the biomagnification and effects of 1,2,3,5,7-pentachloronaphthalene (PeCN52) in an experimental food chain consisting of benthic worms and juvenile rainbow trout. Worms were contaminated with PeCN52 by passive dosing from polydimethylsiloxane silicone. The contaminated worms were then used to feed the juvenile rainbow trout at 0.12, 0.25 or 0.50 μg/g fish wet weight/day, and the resulting internal whole-body concentrations of the individual fish were linked to biological responses. A possible involvement of the cellular detoxification system was explored by measuring PeCN52-induced expression of the phase I biotransformation enzyme gene cyp1a1 and the ABC transporter gene abcb1a. At the end of the 28-day study, biomagnification factors were similar for all dietary intake levels with values between 0.5 and 0.7 kg lipid(fish)/kg lipid(worm). The average uptake efficiency of 60% indicated that a high amount of PeCN52 was transferred from the worms to the fish. Internal concentrations of up to 175 mg/kg fish lipid in the highest treatment level did not result in effects on survival, behavior, or growth of the juvenile trout, but were associated with the induction of phase I metabolism which was evident from the significant up-regulation of cyp1a1 expression in the liver. In contrast, no changes were seen in abcb1a transcript levels.

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

Lipid-dependent topogenesis and function of membrane proteins

Membranes are essential for the integrity and function of the cell. The collective property of the lipid bilayer is critical in providing an optimal functioning environment for membrane proteins. The simple yet well-characterized bacterium Escherichia coli serves an ideal model system to study the function of specific lipids since its lipid content can be easily manipulated. The most abundant lipid in E. coli membrane is phosphatidylethanolamine (PE, 70-80%). A PE-lacking E. coli mutant displays a complex mixture of deficient phenotypes, suggesting a profound role for PE in different aspects of cell function. A novel role of PE as a topological and functional determinant for membrane proteins has been established using lactose permease (LacY) as a model protein. PE is found to be required for energy-dependent uphill transport process of LacY. In PE-lacking membranes, LacY undergoes a dramatic conformational change, and the first half of the protein adopts an inverted topology with respect to the bilayer plane. ^ The work reported here was initiated to understand the molecular properties of lipids that enable their function as topological and functional determinants for membrane proteins. A glycolipid, monoglucosyldiacylglycerol (MGlcDAG) which shares physicochemical similarities with PE, was introduced to PE-lacking E. coli membranes. The introduction of MGlcDAG suppresses many of the PE-deficient phenotypes, and in particular supports the function and native topology of LacY. ^ The lipid-sensitive topogenic signals encoded in the amino acid sequence of LacY were also identified. Native LacY adopts an inverted topology when synthesized without PE, but mutation of specific acidic residues in the cytoplasmic extra-membrane domains can prevent this inversion and supports a native topological organization of LacY in PE-lacking membranes. These results suggest that it is the interplay between the collective charge properties of the lipid bilayer and extra-membrane loops of protein that determines the final orientation of transmembrane domains. By comparing the similarities as well as differences between these two lipids, we established how specific physical and chemical properties of lipids influence various cell functions and elucidated the molecular basis for the novel role of lipids in determining membrane protein topology. ^

Molecular interactions and signal transfer between sensory rhodopsin-I and its cognate transducer

SRI is unique among known photoreceptors in that it produces opposite signals depending on the color of light stimuli. Absorption of orange light (587 nm) triggers an attractant response by the cell, whereas absorption of orange light followed by near-UV light (373 run) triggers a repellent response. Using behavioral mutants that exhibit aberrant color-sensing ability, we tested a two-conformation equilibrium model, using FRET and EPR spectroscopy. The essence of the model applied to SRI-HtrI is that the complex exists in a metastable two-conformer equilibrium which is shifted in one direction by orange light absorption (producing an attractant signal) and in the opposite direction by a second UV-violet photon (producing a repellent signal). First, by FRET we found that the E-F cytoplasmic loop of SRI moves toward the RAMP domain of the HtrI transducer during the formation of the orange-light activated signaling state of the complex. This is the first localization of a change in the physical relationship between the receptor and transducer subunits of the complex and provides a structural property of the two proposed conformers that we can monitor. Second, EPR spectra of a spin label probe at this cytoplasmic position showed shifts in the dark in the mutants toward shorter or longer EF loop-RAMP distances, explaining their behavior in terms of their mutations causing pre-stimulus shifts into one or the other conformer. ^ Next, we applied a novel electrophysiological method for monitoring the directionality of proton movement during photoactivation of SRI, to investigate the process of proton transfer in the photoactive site from the chromophore to proton acceptors on both the wildtype and aberrant color-response mutants. We observed an unexpected and critical difference in the two signaling conformations of the SRI-HtrI complex. The finding is that the vectoriality (i.e. movement away or toward the cytoplasm) of the light-induced proton transfer from the chromophore to the protein is opposite in formation of the two conformations. Retinylidene proton transfer is a common critical process in rhodopsins and these results are the first to show differences in vectoriality in a rhodopsin receptor, and to demonstrate functional importance of the direction of proton transfer. ^

Insulin-like growth factor binding protein 2 (IGFBP2) promotes glioma development and progression

Overexpression of insulin-like growth factor binding protein 2 (IGFBP2) is associated with progression and poor survival in many types of human cancer (such as prostate, ovarian, adrenocortical, breast, colorectal carcinomas, leukemia, and high-grade gliomas). We therefore hypothesize that IGFBP2 is a key regulator of tumor progression. We tested our hypothesis in gliomas using the somatic gene transfer RCAS-tva mouse model system, which permits the introduction of specific genes into specific, cell lineages, in this case glial cells (RCAS: Replication competent avian sarcomavirus, tv-a: avian RCAS virus receptor). Mice are transgenic and harbor the tv-a receptor under the control of a glial-specific promoter and study genes are cloned into the RCAS vector for post-natal intracranial delivery. For these experiments, the study genes were IGFBP2, platelet-derived growth factor B (PDGFB), K-Ras, Akt, and IIp45 (invasion inhibitory protein 45 kDa; known to bind and block IGFBP2 activity), which were delivered separately and in combination. Our results show that PDGFB signaling leads exclusively to the formation of low-grade (WHO grade II) oligodendrogliomas. PDGFB delivered in combination with IGFBP2 results in the formation of anaplastic oligodendrogliomas (WHO grade III), which are characterized by increased cellularity, vascular proliferation, small regions of necrosis, increased mitotic activity, and increased activation of the Akt pathway. IIp45 injected in combination with PDGFB and IGFBP2 ablates IGFBP2-induced tumor progression, which results in formation of low-grade oligodendrogliomas, and an overall reduction in tumor incidence. K-Ras expression was required to form astrocytomas with either IGFBP2 or Akt, indicating the activation of two separate pathways is necessary for gliomagenesis. In ex vivo experiments, blockade of Akt by an inhibitor led to decreased viability of cells co-expressing IGFBP2 versus PDGFB expression alone. This study provides definitive evidence, for the first time, that: (1) IGFBP2 plays a role in activation of the Akt pathway, (2) IGFBP2 collaborates with K-Ras or PDGFB in the development and progression of two major types of glioma, and (3) IGFBP2-induced tumor progression can be ablated by IIp45 or by specific inhibition of the Akt pathway. ^

Contribution of transmembrane and cytoplasmic domains to membrane protein association

Membrane proteins are critical to every aspect of cell physiology, with their association mediating important biological functions. The transmembrane and cytoplasmic domains are known to be important for their association. In order to characterize their role in detail, we have applied different biophysical techniques in detergent micelles to two model systems. The first study involves FcRγ, a single transmembrane domain protein existing as a disulfide linked homodimer. We investigated the role of a conserved transmembrane polar residue and the cytoplasmic tail in FcRγ homo-interactions. Our results by various biophysical techniques including SDS-PAGE, circular dichroism and sedimentation equilibrium in detergent micelles indicate importance of both the transmembrane polar residue and cytoplasmic tail in maintaining proper conformation for FcRγ homo-interactions. A contrasting second study was on L-selectin, another single transmembrane domain protein with a large extracellular domain and a short cytoplasmic tail. Previous cross-linking experiments indicate its possible dimerization. However, the purified fragment of L-selectin and corresponding mutants did not dimerize when analyzed by TOXCAT assay, sedimentation equilibrium and fluorescence resonance energy transfer. It was likely that the presence of juxtamembrane positively charged residues led to decreased migrational rates in SDS PAGE. In conclusion, complementary biophysical techniques should be used with care when studying membrane protein association in detergent micelles. As an extension to our study on L-selectin, we also investigated its interaction with Calmodulin (CaM) in detergent micelles. CaM was found to interact with different detergents. We applied fluorescence and NMR spectroscopy to characterize the interaction of both the apo and Ca 2+ bound form of CaM, with commonly used detergents, below and above their respective critical micelle concentrations. The interaction of apo-CaM with detergents was found to vary with the nature of the detergent head group, whereas Ca2+-CaM interacted with individual detergent molecules irrespective of the nature of their head group. NMR titration experiments of CaM with detergents indicated involvement of specific residues from the N-lobe, linker and C-lobe of CaM. ^

(Table 1) Lipid and organochlorine content in blood of common eiders (Somateria mollissima) in Svalbard

Female common eiders (Somateria mollissima) starve during the nesting stage and may lose 30-45% of their initial body mass, mostly through lipid mobilization. In this study, the effects of fasting on the blood concentrations of three lipid-soluble organochlorines (OCs: polychlorinated biphenyl [PCB]-153; 1-dichloro-2,2-bis (p-chlorophenyl) ethylene [p,p'-DDE]; and hexachlorobenzene [HCB]) were examined in eiders breeding in the high Arctic. Blood samples were taken from females (n = 47) at day 5 and day 20 of the incubation period. The mean wet weight concentrations of PCB-153 and p,p'-DDE increased strongly between day 5 and day 20 (3.6 and 8.2-fold, respectively), while HCB increased less (1.7-fold). There was a strong negative association between daily increase in PCB-153 and clutch size, and a weaker relationship for p,p'-DDE, suggesting that maternal transfer to the eggs is a significant pathway of elimination of OCs in eiders. Moreover, poor body condition (body mass controlled for body size) late in the incubation period was associated with strong daily increase of both p,p'-DDE and PCB-153, which may suggest that the release of these compounds increases when lipid reserves become depleted. For HCB, the increase was mainly associated with increase in blood lipid concentrations, and weakly to the amount of burned lipids. The causes for the differences between the compounds are, however, poorly understood. Although the absolute levels of OCs in eiders were relatively low, their rapid build-up during incubation is worrying as it coincides with poor body condition and weakened immune systems.

Photosynthetically active radiation during the experiment, and lipid content of sub- and intertidal specimens of the endemic Antarctic red alga, Palmaria decipiens

In coastal waters, Antarctic rhodophytes are exposed to harsh environmental conditions throughout the year, like low water temperatures ranging from -1.8°C to 2°C and high light during the summer season. Photosynthetic performance under these conditions may be affected by slowed down enzymatic reactions and the increased generation of reactive oxygen species. The consequence might be a chronic photoinhibition of photosynthetic primary reactions related to increased fragmentation of the D1 reaction centre protein in photosystem II. It is hypothesized that changes in lipid composition of biomembranes may represent an adaptive trait to maintain D1 turnover in response to temperature variation. The interactive effects of high light and low temperature were studied on an endemic Antarctic red alga, Palmaria decipiens, sampled from two shore levels, intertidal and subtidal, and exposed to mesocosm experiments using two levels of natural solar radiation and two different temperature regimes (2-5°C and 5-10°C). During the experimental period of 23 days, maximum quantum yield of photosynthesis decreased in all treatments, with the intertidal specimens exposed at 5-10°C being most affected. On the pigment level, a decreasing ratio of phycobiliproteins to chlorophyll a was found in all treatments. A pronounced decrease in D1 protein concentration occurred in subtidal specimens exposed at 2-5°C. Marked changes in lipid composition, i.e. the ratio of saturated to unsaturated fatty acids, indicated an effective response of specimens to temperature change. Results provide new insights into mechanisms of stress adaptation in this key species of shallow Antarctic benthic communities.

Heterologous Expression of a Plant Small Heat-Shock Protein Enhances Escherichia Coli Viability under Heat And Cold Stress Ref.: 1

A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17.5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37°C to 50°C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4°C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress

Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21cip1. and p16INK4a, and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0°C) and stage B (accumulated at 30°C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.

Identification of the block in targeted retroviral-mediated gene transfer

A chimeric retroviral vector (33E67) containing a CD33-specific single-chain antibody was generated in an attempt to target cells displaying the CD33 surface antigen. The chimeric envelope protein was translated, processed, and incorporated into viral particles as efficiently as wild-type envelope protein. The viral particles carrying the 33E67 envelope protein could bind efficiently to the CD33 receptor on target cells and were internalized, but no gene transfer occurred. A unique experimental approach was used to examine the basis for this postbinding block. Our data indicate that the chimeric envelope protein itself cannot participate in the fusion process, the most reasonable explanation being that this chimeric protein cannot undergo the appropriate conformational change that is thought to be triggered by receptor binding, a suggested prerequisite to subsequent fusion and core entry. These results indicate that the block to gene transfer in this system, and probably in most of the current chimeric retroviral vectors to date, is the inability of the chimeric envelope protein to undergo this obligatory conformational change.

NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone

The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron–sulfur cluster N2 to quinone.

Translocation of inserted foreign epitopes by a channelforming protein

Certain bacterial protein toxins are able to insert themselves into, and at least partially across, lipid bilayer membranes in the absence of any auxiliary proteins, by using unknown mechanisms to overcome the high energy barrier presented by the hydrophobic bilayer core. We have previously shown that one such toxin, colicin Ia, translocates a large, hydrophilic part of itself completely across a lipid bilayer in conjunction with the formation of an ion-conducting channel. To address the question of whether the colicin can translocate any arbitrary amino acid sequence, we have altered the translocated segment by inserting, singly, two different foreign epitopes. Colicins containing either epitope retain significant bactericidal activity and form channels of normal conductance in planar bilayers. Furthermore, antibodies added on the side of the bilayer opposite that to which the colicin was added interact specifically with the corresponding epitopes, producing an inhibition of channel closing. Thus, the inserted epitopes are translocated along with the rest of the segment, suggesting that a surprisingly small part of colicin Ia, located elsewhere in the molecule, acts as a nonspecific protein translocator.

Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology

The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal fluid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of fluorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potential-sensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal fluid.