Studies on the adhesion properties of the liver-metastatic murine RAW117 large-cell lymphoma cells: Roles of $\beta\sb3$ integrin

Metastasis is the major cause of death in cancer patients. Since many cancers show organ-preference of metastasis, elucidation of the underlying mechanisms of metastasis will benefit diagnosis or treatment of metastatic diseases. Adhesion mechanisms are thought to be involved in organ-preference of metastasis, because metastatic cells show organ preference in adhering to organ-derived microvascular endothelial cells. The adhesion molecules in this process remain largely unidentified. I have examined a series of murine RAW117 large-cell lymphoma cells variants selected in vivo for liver-colonizing properties ($\rm{H10{>>}L17>P}$). The highly liver-metastatic H10 cells were found to differentially express much higher levels of integrin $\alpha\rm\sb{v}\beta\sb3$ than L17 or P cells. H10 cells also adhered at higher rates to vitronectin and fibronectin than to fibrinogen, fibrin, laminin and type I collagen, and adhered at significantly higher rates to (GRGDS)$\sb4$ than to monomeric RGD-peptides. In contrast, P and L17 cells did not adhere well to the above substrates. H10 cells also spread well on vitronectin and migrated toward vitronectin concentration gradients. Pretreament of H10 cells with anti-$\beta\sb3$ monoclonal antibodies resulted in significant decreases in adhesion of H10 cells to vitronectin and immobilized (GRGDS)$\sb4$, and reduced the formation of experimental liver metastases in syngeneic Balb/c mice.^ Adhesion of RAW117 cells under hydrodynamic shear stresses was also studied because tumor cell adhesion occurs under fluid shear stresses in target organ microvessels. Similar to their properties found with static adhesion assays, H10 cells stabilized their hydrodynamic adhesion to vitronectin, fibronectin and (GRGDS)$\sb4$ much more quickly than P or L17 cells. Unlike their static adhesion properties, RAW117 cells showed differential adhesion stabilization to liver-sinusoidal endothelial cell-derived extracellular matrix ($\rm{H10{>>}L17>P}$). Although not supporting static adhesion of RAW117 cells, monomeric RGD-peptides mediated adhesion stabilization of H10 cells but not L17 or P cells. Integrin $\rm\alpha\sb{v}\beta\sb3$ was found to be involved in stabilizing H10 cell adhesion to vitronectin, (GRGDS)$\sb4$, monomeric RGD-peptide R1, and liver sinusoidal endothelial cell-derived extracellular matrix.^ This study is the first to provide evidence that integrin $\rm\alpha\sb{v}\beta\sb3$ is differentially expressed in liver-metastatic lymphoma cells and involved in differential adhesion of these cells. The results indicate that strong static adhesion and especially the unique hydrodynamic adhesion of RAW117 cells to the RGD-containing substrates correlate with liver-metastatic potentials. Thus, integrin $\rm\alpha\sb{v}\beta\sb3$ may play an important role in liver-preferential metastasis of RAW117 large-cell lymphoma cells. ^

Multifunctional nanoparticles in cancer: In vitro characterization, in vivo distribution, and cellular response after laser-NIR dye-induced heating

A novel biocompatible and biodegradable polymer, termed poly(Glycerol malate co-dodecanedioate) (PGMD), was prepared by thermal condensation method and used for fabrication of nanoparticles (NPs). PGMD NPs were prepared using the single oil emulsion technique and loaded with an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The size of the void PGMD NPs, IR820-PGMD NPs and DOX-IR820-PGMD NPs were approximately 90 nm, 110 nm, and 125 nm respectively. An acidic environment (pH=5.0) induced higher DOX and IR820 release compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of DOX-IR820-PGMD NPs was comparable in MES-SA but was higher in Dx5 cells compared to free DOX plus IR820 (p<0.05). The combination of hyperthermia (HT) and chemotherapy improved cytotoxicity in both cell lines. We also explored the cellular response after rapid, short-term and low thermal dose (laser/Dye/NP) induced-heating, and compared it to slow, long-term and high thermal dose cell incubator heating by investigating the reactive oxygen species (ROS) level, hypoxia-inducible factor-1&agr; (HIF-1&agr;) and vascular endothelial growth factor (VEGF) expression. The cytotoxicity of IR820-PGMD NPs after laser/Dye/NP HT resulted in higher cancer cell killing compared to incubator HT. ROS level, HIF-1&agr; and VEGF expression were elevated under incubator HT, while maintained at the baseline level under the laser/Dye/NP HT. In vivo mouse studies showed that NP formulation significantly improved the plasma half-life of IR820 after tail vein injection. Significant lower IR820 content was observed in kidney in DOX-IR820-PGMD NP treatment as compared to free IR820 treatment in our biodistribution studies (p<0.05). In conclusion, both IR820-PGMD NPs and DOX-IR820-PGMD NPs were successfully developed and used for both imaging and therapeutic purposes. Rapid and short-term laser/Dye/NP HT, with a low thermal dose, did not up-regulate HIF-1&agr; and VEGF expression, whereas slow and long-term incubator HT, with a high thermal dose, can enhance expression of both HIF-1&agr; and VEGF.^

Improved antiretroviral treatment outcome in a rural African setting is associated with cART initiation at higher CD4 cell counts and better general health condition

Background Data on combination antiretroviral therapy (cART) in remote rural African regions is increasing. Methods We assessed prospectively initial cART in HIV-infected adults treated from 2005 to 2008 at St. Francis Designated District Hospital, Ifakara, Tanzania. Adherence was assisted by personal adherence supporters. We estimated risk factors of death or loss to follow-up by Cox regression during the first 12 months of cART. Results Overall, 1,463 individuals initiated cART, which was nevirapine-based in 84.6%. The median age was 40 years (IQR 34-47), 35.4% were males, 7.6% had proven tuberculosis. Median CD4 cell count was 131 cells/μl and 24.8% had WHO stage 4. Median CD4 cell count increased by 61 and 130 cells/μl after 6 and 12 months, respectively. 215 (14.7%) patients modified their treatment, mostly due to toxicity (56%), in particular polyneuropathy and anemia. Overall, 129 patients died (8.8%) and 189 (12.9%) were lost to follow-up. In a multivariate analysis, low CD4 cells at starting cART were associated with poorer survival and loss to follow-up (HR 1.77, 95% CI 1.15-2.75, p = 0.009; for CD4 <50 compared to >100 cells/μl). Higher weight was strongly associated with better survival (HR 0.63, 95% CI 0.51-0.76, p < 0.001 per 10 kg increase). Conclusions cART initiation at higher CD4 cell counts and better general health condition reduces HIV related mortality in a rural African setting. Efforts must be made to promote earlier HIV diagnosis to start cART timely. More research is needed to evaluate effective strategies to follow cART at a peripheral level with limited technical possibilities.

Signal relay between two integral membrane proteins: The sensory rhodopsin I/HtrI transducer molecular complex

The molecular complex of sensory rhodopsin I (SRI) and its transducer HtrI mediate color-sensitive phototaxis in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light causes a repellent response by a two-photon reaction. Three aspects of this molecular complex were explored: (i) We determined the stoichiometry of SRI and HtrI to be 2:2 by gene fusion analysis. A SRI-HtrI fusion protein was expressed in H. salinarum and shown to mediate 1-photon and 2-photon phototaxis responses comparable to wild-type complex. Disulfide crosslinking demonstrated that the fusion protein is a homodimer in the membrane. Measurement of photochemical reaction kinetics and pH titration of absorption spectra established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. (ii) Cytoplasmic channel closure of SRI by HtrI, an important aspect of their interaction, was investigated by incremental HtrI truncation. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. The closure activity is localized to 5 specific residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp76 counterion to the protonated Schiff base chromophore. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. (iii) We developed a procedure for reconstituting HtrI-free SRI and the SRI/HtrI complex into liposomes, which exhibit photocycles with opened and closed cytoplasmic channels, respectively, as in the membrane. This opens the way for study of the light-induced conformational change and the interaction in vitro by fluorescence and spin-labeling. Single-cysteine mutations were introduced into helix F of SRI, labeled with a nitroxide spin probe and a fluorescence probe, reconstituted into proteoliposomes, and light-induced conformational changes detected in the complex. The probe signals can now be used as the readout of signaling to analyze mutants and the kinetics of signal relay. ^

Spectroscopic and functional investigations of the AMPA subtype of ionotropic glutamate receptors

Ion channels play a crucial role in the functioning of different systems of the body because of their ability to bridge the cell membrane and allow ions to pass in and out of the cell. Ionotropic glutamate receptors are one class of these important proteins and have been shown to be critical in propagating synaptic transmission in the central nervous system and in other diverse functions throughout the body. Because of their wide-ranging effects, this family of receptors is an important target for structure-function investigations to understand their mechanism of action. ^ α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are one subtype of glutamate receptors and have been shown to be the primary receptors involved in rapid excitatory signaling in the central nervous system. Agonist binding to the extracellular ligand binding domain of these receptors causes various conformational changes that culminate in formation of the ion channel. Previous structural investigations have provided important information about their mechanism of action, including uncovering a relationship between the degree of cleft closure in the binding domain and activation of the receptor. However, what question remains unanswered is how specific interactions between the agonist and the protein interplay with cleft closure to mediate receptor activation. ^ To investigate this question, I applied a multiscale approach to investigate the effects of agonist binding on various levels. Vibrational spectroscopy was utilized to investigate molecular-level interactions in the binding pocket, and fluorescence resonance energy transfer (FRET) was employed to measure cleft closure in the isolated ligand binding domain. The results of these studies in the isolated binding domain were then correlated to activation of the full receptor. These investigations showed a relationship between the strength of the interaction at the α-amine group of the agonist and extent of receptor activation, where a stronger interaction correlated to a larger activation, which was upheld even when the extent of cleft closure did not correlate to activation. These results show that this interaction at the α-amine group is critical in mediating the allosteric mechanism of activation and provide a bit more insight into how agonist binding is coupled to channel gating in AMPA receptors. ^

Evidence for the role of agonist binding frequency in receptor -mediated activation of adenylate cyclase

$\beta$-adrenergic receptor-mediated activation of adenylate cyclase exhibits an agonist-specific separation between the dose/response curve (characterized by the EC$\sb{50}$) and the dose/binding curve (characterized by the K$\sb{\rm d}$). Cyclase activity can be near-maximal when receptor occupancy is quite low (EC$\sb{50}$ $\ll$ K$\sb{\rm d}$). This separation between the binding and response curves can be explained by the assumption that the rate of cyclase activation is proportional to the concentration of agonist-bound receptors, since the receptor is mobile and can activate more than one cyclase (the Collision Coupling Model of Tolkovsky and Levitzki). Here it is established that agonist binding frequency plays an additional role in adenylate cyclase activation in S49 murine lymphoma cells. Using epinephrine (EC$\sb{50}$ = 10 nM, K$\sb{\rm d}$ = 2 $\mu$M), the rate of cyclase activation decreased by 80% when a small (1.5%) receptor occupancy was restricted (by addition of the antagonist propranolol) to a small number (1.5%) of receptors rather than being proportionally distributed among the cell's entire population of receptors. Thus adenylate cyclase activity is not proportional to receptor occupancy in all circumstances. Collisions between receptor and cyclase pairs apparently occur a number of times in rapid sequence (an encounter); the high binding frequency of epinephrine ensures that discontiguous regions of the cell surface experience some period of agonist-bound receptor activity per small unit time minimizing "wasted" collisions between activated cyclase and bound receptor within an encounter. A contribution of agonist binding frequency to activation is thus possible when: (1) the mean lifetime of the agonist-receptor complex is shorter than the mean encounter time, and (2) the absolute efficiency (intrinsic ability to promote cyclase activation per collision) of the agonist-receptor complex is high. These conclusions are supported by experiments using agonists of different efficiencies and binding frequencies. These results are formalized in the Encounter Coupling Model of adenylate cyclase activation, which takes into explicit account the agonist binding frequency, agonist affinity for the $\beta$-adrenergic receptor, agonist efficiency, encounter frequency and the encounter time between receptor and cyclase. ^

The effects of ultraviolet B radiation on immune responses to {\it Borrelia burgdorferi}, the causative agent of Lyme disease

Ultraviolet B (UVB) radiation, in addition to being carcinogenic, is also immunosuppressive. Immunologically, UVB induces suppression locally, at the site of irradiation, or systemically, by inducing the production of a variety of immunosuppressive cytokines. Systemic effects include suppression of delayed-type hypersensitivity (DTH) responses to a variety of antigens (e.g. haptens, proteins, bacterial antigens, or alloantigens). One of the principal mediators of UV-induced immune suppression is the T helper-2 (Th2) cytokine interleukin-10 (IL-10); this suggests that UV irradiation induces suppression by shifting the immune response from a Th1 (cellular) to a Th2 (humoral) response. These "opposing" T helper responses are usually mutually exclusive, and polarized Th1 or Th2 responses may lead to either protection from infection or increased susceptibility to disease, depending on the infectious agent and the route of infection.^ This study examines the effects of UVB irradiation on cellular and humoral responses to Borrelia burgdorferi (Bb), the causative agent of Lyme disease (LD) in both immunization and infectious disease models; in addition, it examines the role of T cells in protection from and pathology of Bb infection. Particular emphasis is placed on the Bb-specific antibody responses following irradiation since UVB effects on humoral immunity are not fully understood. Mice were irradiated with a single dose of UV and then immunized (in complete Freund's adjuvant) or infected with Bb (intradermally at the base of the tail) in order to examine both DTH and antibody responses in both systems. UVB suppressed the Th1-associated antibodies IgG2a and IgG2b in both systems, as well as the DTH response to Bb in a dose dependent manner. Injection of anti-IL-10 antibody into UV-irradiated mice within 24 h after UV exposure restored the DTH response, as well as the Th1 antibody (IgG2a and IgG2b) response. In addition, injecting recombinant IL-10 mimicked some of the effects of UV radiation.^ Bb-specific Th1 T cell lines (BAT2.1-2.3) were generated to examine the role of T cells in Lyme borreliosis. All lines were CD4$\sp+,$ $\alpha\beta\sp+$ and proliferated specifically in response to Bb. The BAT2 cell lines not only conferred a DTH response to naive C3H recipients, but reduced the number of organisms recovered from the blood and tissues of mice infected with Bb. Furthermore, BAT2 cell lines protected mice from Bb-induced periarthritis. ^

NMR structural analysis of cardiac troponin C: Monitoring conformational changes induced by binding calcium, troponin I, and calcium -sensitizing drugs

Contraction of cardiac muscle is regulated through the Ca2+ dependent protein-protein interactions of the troponin complex (Tn). The critical role cardiac troponin C (cTnC) plays as the Ca2+ receptor in this complex makes it an attractive target for positive inotropic compounds. In this study, the ten Met methyl groups in cTnC, [98% 13C ϵ]-Met cTnC, are used as structural markers to monitor conformational changes in cTnC and identify sites of interaction between cTnC and cardiac troponin I (cTnI) responsible for the Ca2+ dependent interactions. In addition the structural consequences that a number of Ca2+-sensitizing compounds have on free cTnC and the cTnC·cTnI complex were characterized. Using heteronuclear NMR experiments and monitoring chemical shift changes in the ten Met methyl 1H-13C correlations in 3Ca2+ cTnC when bound to cTnI revealed an anti-parallel arrangement for the two proteins such that the N-domain of cTnI interacts with the C-domain of cTnC. The large chemical shifts in Mets-81, -120, and -157 identified points of contact between the proteins that include the C-domain hydrophobic surface in cTnC and the A, B, and D helical interface located in the regulatory N-domain of cTnC. TnI association [cTnI(33–80), cTnI(86–211), or cTnI(33–211)] was found also to dramatically reduce flexibility in the D/E central linker of cTnC as monitored by line broadening in the Met 1H- 13C correlations of cTnC induced by a nitroxide spin label, MTSSL, covalently attached to cTnC at Cys 84. TnI association resulted in an extended cTnC that is unlike the compact structure observed for free cTnC. The Met 1H-13C correlations also allowed the binding characteristics of bepridil, TFP, levosimendan, and EMD 57033 to the apo, 2Ca2+, and Ca2+ saturated forms of cTnC to be determined. In addition, the location of drug binding on the 3Ca2+cTnC·cTnI complex was identified for bepridil and TFP. Use of a novel spin-labeled phenothiazine, and detection of isotope filtered NOEs, allowed identification of drug binding sites in the shallow hydrophobic cup in the C-terminal domain, and on two hydrophobic surfaces on N-regulatory domain in free 3Ca2+ cTnC. In contrast, only one N-domain drug binding site exists in 3Ca2+ cTnC·cTnI complex. The methyl groups of Met 45, 60 and 80, which are grouped in a hydrophobic patch near site II in cTnC, showed the greatest change upon titration with bepridil or TFP, suggesting that this is a critical site of drug binding in both free cTnC and when associated with cTnI. The strongest NOEs were seen for Met-60 and -80, which are located on helices C and D, respectively, of Ca2+ binding site II. These results support the conclusion that the small hydrophobic patch which includes Met-45, -60, and -80 constitutes a drug binding site, and that binding drugs to this site will lead to an increase in Ca2+ binding affinity of site II while preserving maximal cTnC activity. Thus, the subregion in cTnC makes a likely target against which to design new and selective Ca2+-sensitizing compounds. ^

Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating phosphatidylinositol 3 kinase localization in Dictyostelium discoideum

Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. ^ It was initially found that comparing to wild type cells, gsk3 - cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. ^ I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.^

Molecular analysis of cell surface $\beta$1,4-galactosyltransferase function during lamellipodal formation, cell polarization, and cell migration

The rate and direction of fibroblast locomotion is regulated by the formation of lamellipodia. In turn, lamellipodal formation is modulated in part by adhesion of that region of the cell from which the lamellipodia will extend or orginate. Cell surface $\beta$1,4-galactosyltransferase (GalTase) is one molecule that has been demonstrated to mediate cellular interactions with extracellular matrices. In the case of fibroblasts, GalTase must be associated with the actin cytoskeleton in order to mediate cellular adhesion to laminin. The object of this study was to determine how altering the quantity of GalTase capable of associating with the cytoskeleton impacts cell motility. Stably transfected cell lines were generated that have increased or decreased levels of surface GalTase relative to its cytoskeleton-binding sites. Biochemical analyses of these cells reveals that there is a limited number of sites on the cytoskeleton with which GalTase can interact. Altering the ratio of GalTase to its cytoskeleton binding sites does not affect the cells' abilities to spread, nor does it affect the localization of cytoskeletally-bound GalTase. It does, however, appear to interfere with stress fiber bundling. Cells with altered GalTase:cytoskeleton ratios change their polarity of laminin more frequently, as compared to controls. Therefore, the ectopic expression of GalTase cytoplasmic domains impairs a cell's ability to control the placement of lamellipodia. Cells were then tested for their ability to respond to a directional stimulus, a gradient of platelet-derived growth factor (PDGF). It was found that the ability of a cell to polarize in response to a gradient of PDGF is directly proportional to the quantity of GalTase associated with its cytoskeleton. Finally, the rate of unidirectional cell migration on laminin was found to be directly dependent upon surface GalTase expression and is inversely related to the ability of surface GalTase to interact with the cytoskeleton. It is therefore proposed that cytoskeletal assembly and lamellipodal formation can be regulated by the altering the ratio of cytoplasmic domains for specific matrix receptors, such as GalTase, relative to their cytoskeleton-binding sites. ^

Structure and function study of prostaglandin H synthase

Prostaglandin H synthase (PGHS) is a key enzyme in biosynthesis of prostaglandins, thromboxane, and prostacyclin. It has two activities, cyclooxygenase and peroxidase. "PGHS" means PGHS-1. A current hypothesis considers the cyclooxygenase reaction to be a free radical chain reaction, initiated by interaction of the synthase peroxidase with hydroperoxides leading to the production of a tyrosyl free radical. According to this hypothesis, tyrosyl residue(s) may play a key role in the cyclooxygenase reaction. Tetranitromethane (TNM) can relatively selectively nitrate tyrosines at pH 8.0. The effect of TNM on both cyclooxygenase activity and peroxidase activity has been examined: reaction of the synthase holoenzyme with TNM at pH 8.0 led to inactivation of both activities, with the cyclooxygenase activity being lost rapidly and completely, while the peroxidase activity was lost more slowly. Indomethacin, a non-steroidal anti-inflammatory agent, can protect the synthase from the inactivation of TNM. Amino acid analyses indicated that a loss of tyrosine and formation of nitrotyrosine residues occurred during reaction with TNM, and that TNM-reacted holoenzyme with $<$10% residual cyclooxygenase activity had about 2.0 nitrotyrosine/subunit.^ PGH synthase is known to be an endoplasmic reticulum membrane-associated protein. Antibodies directed at particular PGHS peptide segments and indirect immunofluorescence have been used to characterize the membrane topology of crucial portions of PGHS. PGHS was expressed in COS-1 cells transfected with the appropriate cDNA. Stably-transfected human endothelial cells were also used for the topology study. The cells were treated with streptolysin-O, which selectively permeabilizes the plasma membrane, or with saponin to achieve general membrane disruption, before incubation with the antipeptide antibodies. Bound antipeptide antibody was stained by FITC-labelled secondary antibody and visualized by fluorescence microscopy. With the antipeptide antibodies against residues 51-66, 156-170 or 377-390, there was a significant reticular and perinuclear pattern of staining in cells permeabilized with saponin but not in cells permeabilized with SLO alone. Antibodies directed against the endogenous C-terminal peptide or against residues 271-284 produced staining in cells permeabilized with saponin, and also in a lower, but significant fraction of cells permeabilized with SLO. Similar results were obtained when COS-1 cells expressing recombinant PGHS with a viral reporter peptide inserted at the C-terminus were stained with antibody against the reporter epitope.^ The PGHS C-terminal sequence is similar to that of the consensus KDEL ER retention signal. The potential function of the PGHS C-terminus segment in ER retention was examined by mutating this segment and analyzing the subcellular distribution of the mutants expressed in COS-1 cells. None of the mutants had an altered subcellular distribution, although some had greatly diminished the enzyme activities. (Abstract shortened by UMI.) ^

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. ^

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. ^

O6-Methylguanine-DNA-Methyltransferase methylation: prevalence and predictive value in head and neck squamous cell carcinoma

Introduction: Le gène O6-méthylguanine-ADN méthyltransferase (MGMT) code pour une enzyme spécifique réparatrice de l’ADN qui protège les cellules de la toxicité des agents alkylants. Ainsi, l’activité du MGMT est un mécanisme majeur de résistance aux agents alkylants. Il a été démontré qu’une diminution de l’expression du gène MGMT par une hyperméthylation du promoteur résulte en une amélioration de la survie chez les patients avec certains types de tumeurs qui sont traitées avec des agents chimiothérapeuthique alkylants. Objectifs: Déterminer la prévalence de la méthylation du gène MGMT chez des patients avec des cancers épidermoïdes localement avancés de la sphère ORL traités avec chimioradiothérapie et évaluer l’impact de cette méthylation sur la survie. Méthodes: Sur 428 patients consécutifs, traités avec chimioradiothérapie à notre institution et suivis pour un période médiane de 37 mois, 199 spécimens chirurgicaux paraffinés ont été récupérés. L’ADN était extrait et modifié par le traitement au bisulfite. Une réaction en chaîne de la polymérase, spécifique à la méthylation était entreprise pour évaluer l’état de méthylation du promoteur du gène du MGMT. Les résultats de laboratoire étaient corrélés avec la réponse clinique. L’analyse statistique était exécutée à l’aide du test de Fisher pour les données catégoriques et à l’aide des courbes de Kaplan-Meier pour les échecs au traitement. Résultats : Des 199 extraits d’ADN initiaux, 173 (87%) étaient modifiés au bisulfite avec succès. Des ces spécimens modifiés, 71 (41%) ont démontré une hyperméthylation du MGMT. Pour les cas de méthylation et nonméthylation du MGMT, les caractéristiques des patients n’étaient pas significativement différentes. Les taux de réponse étaient 71 et 73% (p=NS) respectivement. Le contrôle locorégional était respectivement 87 et 77% (p=0.26), la survie sans maladie était 80 et 60% (p=0.38), la survie sans métastase à distance était 92 et 78% (p=0.08) et la survie globale était 64 et 62% (p=0.99) à 3 ans. Conclusions : L’état de méthylation du MGMT est fortement prévalent (41%) et semble avoir un possible impact bénéfique sur la survie quand la chimioradiothérapie est administrée aux patients avec des stades avancés de cancers tête et cou.

Inherited and noninherited sources of variation in metastasis and the growth rate of tumors: Implications for tumor evolution and therapy

I have undertaken measurements of the genetic (or inherited) and nongenetic (or noninherited) components of the variability of metastasis formation and tumor diameter doubling time in more than 100 metastatic lines from each of three murine tumors (sarcoma SANH, sarcoma SA4020, and hepatocarcinoma HCA-I) syngeneic to C3Hf/Kam mice. These lines were isolated twice from lung metastases and analysed immediately thereafter to obtain the variance to spontaneous lung metastasis and tumor diameter doubling time. Additional studies utilized cells obtained from within 4 passages of isolation. Under the assumption that no genetic differences in metastasis formation or diameter doubling time existed among the cells of a given line, the variance within a line would estimate nongenetic variation. The variability derived from differences between lines would represent genetic origin. The estimates of the genetic contribution to the variation of metastasis and tumor diameter doubling time were significantly greater than zero, but only in the metastatic lines of tumor SANH was genetic variation the major source of metastatic variability (contributing 53% of the variability). In the tumor cell lines of SA4020 and HCA-I, however, the contribution of nongenetic factors predominated over genetic factors in the variability of the number of metastasis and tumor diameter doubling time. A number of other parameters examined, such as DNA content, karyotype, and selection and variance analysis with passage in vivo, indicated that genetic differences existed within the cell lines and that these differences were probably created by genetic instability. The mean metastatic propensity of the lines may have increased somewhat during their isolation and isotransplantation, but the variance was only slightly affected, if at all. Analysis of the DNA profiles of the metastatic lines of SA4020 and HCA-I revealed differences between these lines and their primary parent tumors, but not among the SANH lines and their parent tumor. Furthermore, there was a direct correlation between the extent of genetic influence on metastasis formation and the ability of the tumor cells to develop resistance to cisplatinum. Thus although nongenetic factors might predominate in contributing to metastasis formation, it is probably genetic variation and genetic instability that cause the progression of tumor cells to a more metastatic phenotype and leads to the emergence of drug resistance. ^